EA044676B1 - NUCLEIC ACID CONSTRUCTION FOR GENE EXPRESSION IN VITRO AND IN VIVO - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to the field of recombinant production of biological molecules in host cells. The invention relates to nucleotide constructs that allow modification of the expression of a desired gene using both in vitro and in vivo gene expression systems. These constructs can be advantageously used to produce a variety of biological molecules recombinantly on an industrial scale, such as human milk oligosaccharides (HMOs).

State of the art

The commercial importance of bacterial cells for the production of recombinant molecules is increasing. Currently, plasmid expression systems are mainly used for the production of recombinant proteins in bacterial hosts, in particular E. coli. Because these systems provide high gene content and are well known, they have become widespread, also due to the simplicity of the available cloning protocols. However, the use of plasmid-based expression systems, especially on a production scale, also has several disadvantages.

Plasmid-based prokaryotic expression systems are typically characterized by high plasmid copy numbers, for example up to several hundred per cell. Expression plasmids typically carry a gene of interest under the control of a promoter, an origin of replication (ori), and a marker gene for selecting clones carrying the plasmid. In addition, these plasmids (ie vectors) often contain coding or non-coding or non-functional framework sequences. The presence of plasmids and the associated replication machinery alters host cell metabolism (DiazRizzi and Hernandez (2000), Crit. Rev. Biotechnol., 20(2):79-108) and imposes a high metabolic burden on cells, thereby limiting their resources for production recombinant protein. In addition, the use of strong promoters in combination with a high content of gene material gives a rate of recombinant protein production that is usually too high for the host cell, and therefore can lead to rapid and irreversible disruption of cellular metabolism. Consequently, the potential of host cells cannot be fully exploited in plasmid-based systems, resulting in low yield and quality of recombinant protein. Thus, one of the major disadvantages of plasmid-based expression systems can be attributed to the increased nutrient and energy requirements that are required for plasmid replication and maintenance.

Another common phenomenon in plasmid-based systems is the change in plasmid copy number during culture. Recombinant protein production at high expression rates is accompanied by starvation and cellular stress, which lead to an increase in pools of unloaded tRNA. This results in interference with the plasmid copy number (PCN) control mechanism. As a result, the PCN increases rapidly and causes disruption of the culture process (the so-called escape effect).

Segregation instability (ie, the emergence of plasmid-free host cells) and structural instability (ie, mutations in the plasmid sequence) are additional problems often observed in plasmid-based systems. During cell division, cells may lose the plasmid and therefore also the gene of interest. This loss of plasmid depends on several external factors and increases with the number of cell divisions (generations). This means that plasmid-based fermentation is limited by the number of generations or cell doublings.

In general, these properties of plasmid expression systems limit the yield of recombinant protein and reduce the controllability and efficiency of the process.

In the search for an effective alternative to plasmid expression, WO 1996/40722 on genomic expression describes a method that uses the integration of a circular vector (called a circular chromosomal transfer DNA, CTD) containing a selectable marker into a bacterial chromosome (i.e. to the site attB E. coli)). In this method, using duplicated DNA sequences flanking a selection marker, amplification of chromosomal gene content was achieved. Thus, the resulting chromosomal gene content was approximately 15-40 copies per cell, which was similar to the amount achieved with traditionally used plasmid vectors. Cultivation of clones containing carrier DNA into the chromosome integrated into the bacterial genome provided levels of recombinant proteins similar to those obtained using plasmid-based systems (Olson et al., 1998). This method required in vitro ligation of the CTD and was relatively restricted to the attB site for insertion.

Genomic expression systems appear to have great potential for providing stable expression of recombinant genes without selection markers. However, often the expression of a recombinant gene on an industrial scale is achievable only by increasing the content of gene copies in the chromosome to the copy level of the plasmid, since one copy of the gene is not capable of ensuring expression on an industrial scale. In addition, selection of the integration site is a challenge, and regulation of expression is often complex and/or not suitable for industrial production. Thus, there is no simple and efficient genome-based bacterial expression system for pro

- 1 044676 industrial production of recombinant polypeptides.

One approach to overcoming the problem of insufficient production and complex regulation of bacterial genomic expression of heterologous polypeptides is the use of strong inducible promoters to control the transcription of integrated recombinant genes. A number of different inducible promoters have been described. For example, promoters inducible by heat such as λP _R and λP _L , tryptophan depletion such as tip, l -arabinose such as araBAD, mannitol such as mtsE, phosphate starvation such as phoA, nalidixic acid such as recA osmolarity such as proU, glucose depletion such as cst-1, tetracycline such as tetA, pH such as cadA, anaerobic conditions such as nar, T4 infection such as T4 phage gene 32, alkyl or halogen benzoates, such as Pm, alkyl or halotoluenes such as Pu, salicylates such as Psal, and oxygen such as VHb have all been explored as alternatives to IPTG inducible promoters (see, for example, Makrides, SC (1996), Microbiol. Rev., 60, 512-538; Hannig G. & Makrides, S. C. (1998), TIBTECH, 16, 54-60; Stevens, R. C. (2000), Structures, 8, R177-R185; Hoffmann J. & Altenbuchner J. (2015), PLoS One, 10(7):e0133248; J. Sanchez-Romero & V. De Lorenzo, Genetic Engineering of Nonpathogenic Pseudomonas strains as Biocatalysts for Industrial and Environmental Processes, in Manual of Industrial Microbiology and Biotechnology (A. Demain & J. Davies, eds.), p. 460-74 (1999) (ASM Press, Washington, DC); H. Schweizer, Vectors to express foreign genes and techniques to monitor gene expression for Pseudomonas, Current Opinion in Biotechnology, 12:439-445 (2001); R. Slater & R. Williams, The Expression of Foreign DNA in Bacteria, in Molecular Biology and Biotechnology (J. Walker & R. Rapley, eds.), p. 125-54 (2000) (The Royal Society of Chemistry, Cambridge, UK). However, these inducible promoters have a number of problems, such as high temperature induction, which is harmful to cells and may eventually become impractical for large-scale fermentation due to equipment limitations; manipulation of oxygen can affect the overall dynamics of aspects of cell growth density during fermentation, reducing ideal conditions; the use of toluenes or other similar types of potentially toxic chemicals may require additional purification to ensure that these compounds are not present in the final product; and pH can affect the ability of the peptide of interest to fold correctly or dissolve in the host cell, making purification more costly and time-consuming, making plasmid expression systems still the preferred choice for industrial production.

Regulation of promoter activity by a carbon source is probably the most attractive option for controlling the expression of a target polypeptide in an industrial setting. There are several reasons for this, such as more efficient use of the carbon source and reduction of long-term metabolic stress on the host cell. However, at present the choice of such promoters is quite limited, and most of them have been adapted for plasmid expression (Terpe K. Appl. Microbiol. Biotechnol. (2006), 72:211-222). However, the genome of a bacterial cell such as E. coli contains thousands of promoters, and many of them are regulated by changes in the carbon source, allowing the availability of carbon in the environment to influence the expression patterns of the genes under their control. It has been suggested that the global transcriptional regulator, cAMP-CRP, which is formed when glucose levels decrease, regulates at least 378 promoters in the bacterial cell (Shimada T. et al., PloS One, 6(6):e20081 (2011)), however, there is no data to suggest which of these promoters are powerful, capable of ensuring stable, controlled genomic production of recombinant polypeptides in high yield under industrial conditions.

Respiratory glycerol metabolism in Escherichia coli (E. coli) is controlled by 12 genes organized into 5 glp operons: glpFKX, glpABC, glpTQ, glpD, and PglpEGR. The glpFKX operon genes encode glycerol diffusion mediator, glycerol kinase, and fructose 1,6-bisphosphatase. The glpABC operon gene encodes the subunits of anaerobic glycerol-3-phosphate dehydrogenase - A, B and C. The glpTQ operon genes encode the glycerol-3-phosphate carrier and glycerophosphodiesterase. The glpD gene encodes an aerobic glycerol-3-phosphate dehydrogenase. The glpR gene encodes the transcriptional repressor GlpR, glpE a thiosulfate sulfur transferase, and glpG a serine protease. Transcription of the genes of each of the glp operons is controlled by the promoters pglpFKX, pglpABC, pglpTQ, pglpD and pglpEGR, respectively, the activity of which is strictly catabolically regulated (Larson T.J. et al. (1987), Biol. Chem., 262:15869-74; Zhao N. et al. al (1994), J. Bacteriol., 176:2393-239). Recently, Selivano L. et al. (Microb. Cell Fact., 15:28 (2016)) in search of new promoters suitable for the expression of recombinant genes, tested the glp promoter, pglpQ, from Streptomyces coelicolor in a bacterial plasmid expression system, however, this promoter showed rather low activity and therefore was excluded as a promising candidate for industrial application.

The essence of the invention

A first aspect of the invention relates to a nucleotide construct comprising a synthetic non-coding DNA sequence (i) which comprises a first DNA fragment and a second DNA fragment, wherein the first DNA fragment is a DNA sequence derived from the 5' untranslated region (5'-UTR) of glp -gene from Escherichia coli, and the second DNA fragment represents

- 2 044676 combat DNA sequence CAAGGAGGAAACAGCT (SEQ ID NO: 10) or a variant of the specified sequence, and where the first fragment is located before the second fragment. In particular, the inventions relate to a nucleotide construct containing three operably linked DNA sequences: a promoter DNA sequence (ii), a synthetic non-coding DNA sequence containing a ribosome binding site (RBS) (i), and a coding DNA sequence (iii) . Preferably, the first DNA fragment is derived from the 5'UTR DNA sequence of the glpF, glpA or glpD genes and contains the first 5-65 consecutive nucleotides after the transcription start site in the glpF, glpA or glpD promoters. In one preferred embodiment, the promoter DNA sequence (ii) corresponds to a DNA sequence from the glp operon promoter from Escherichia coli (E. coli), for example from the glpFKX, glpABC, glpTQ or glpD operons.

In a second aspect, the invention relates to a recombinant cell, preferably a bacterial recombinant cell, containing a nucleotide construct of the invention.

In a third aspect, the invention relates to an expression system comprising a construct of the invention or a recombinant cell of the invention.

In a fourth aspect, the invention relates to a method for producing one or more biological molecules, eg, a protein, nucleic acid, oligosaccharide, etc., using a construct of the invention and/or a recombinant cell of the invention.

These and other aspects of the invention are described in detail below.

Brief description of drawings

In fig. 1 shows (A) expression levels of a reporter gene (lacZ) from nucleotide constructs containing seventeen different promoter elements combined with SEQ ID NO: 10; each expression cassette containing one promoter also contains a native 5'UTR sequence (after the promoter and before SEQ ID NO: 10) from a gene that is naturally transcribed from the promoter construct;

(B) lacZ expression levels from expression cassettes (A) containing selected promoters PglpT, PglpA and PglpF (shaded bars; b) compared with lacZ expression levels from expression cassettes containing the promoter and a DNA fragment of the original 5'UTR of the corresponding gene, those. glpT, glpA and glpF (open bars; a).

Data show the level of expressed β-galactosidase activity in host cells. Activity was measured in Miller units (units/OD/ml/min).

In fig. 2 shows (A) a schematic representation of an embodiment of a nucleotide construct according to the invention;

(B) a nucleic acid construct of the invention comprising a glpF promoter DNA sequence (SEQ ID NO: 54), synthetic DNA (i) comprising SEQ ID NO: 36 and SEQ ID NO: 10.

The following structural features are indicated:

the transcription initiation site at position +1 is shown in bold;

the RNA polymerase binding site containing boxes -10 and -35 is shown in bold;

four operator sites, OR1, OR2, OR3 and OR4, for binding the transcriptional repressor protein GlpR are highlighted in gray;

two operator sites, CRP1 and CRP2, for binding the transcription activator protein CRP are highlighted with a frame;

a synthetic DNA sequence comprising a 54-nucleotide fragment of the 5'UTR of the glpF gene (in dotted frame) (SEQ ID NO: 36) and SEQ ID NO: 10 (underlined).

In fig. Figure 3 schematically shows the structure of glp promoters (A) PglpA, (B) PglpD, (C) PglpF and (D) PglpT.

The relative positions of the -35 and -10 regions recognized by RNA polymerase are shown as black boxes. The relative positions of cAMP-CRP, GlpR, FNR, and FIS binding sites involved in transcriptional regulation of promoter elements are indicated by small empty arrows; the relative position of the 16-nucleotide fragment of the 5′UTR containing the ribosome binding site (SD) is indicated by small filled arrows.

In fig. 4 presents data demonstrating the level of expression of the lacZ gene expressed in E. coli from one copy of a genome-integrated expression cassette containing (a) DNA fragments corresponding to the original glpF, glpA or glpT promoter sequences, and DNA fragments of the 5'UTR from glpF, glpA or glpT containing a (native) ribosome binding site (RSB) (SEQ ID NO: 57, 56 and 55, respectively) (open bars);

(b) DNA fragments containing the original glpF, glpA or glpT promoter sequences, and 5'UTR DNA fragments from the original glpF, glpA or glpT that lack the native RBS (SEQ ID NO: 1, 2 and 4, respectively), but associated with SEQ ID NO: 10 (shaded bars);

(c) DNA fragments containing the original promoter sequences of glpF, glpA or glpT (SEQ ID NO: 54, 48 and 49, respectively), (each) operably linked to a 54 nucleotide DNA fragment of the 5'UTR of glpF (SEQ ID NO: 36) and further associated with SEQ ID NO: 10 (shaded

- 3 044676 cross stitches).

Data show the level of expressed β-galactosidase activity in host cells. Activity is measured in Miller units (units/OD/ml/min).

In fig. 5 shows data demonstrating catabolic repression of PglpF (SEQ ID NO: 12) when operably linked to the lacZ reporter gene integrated (in a single copy) into the E. coli genome. β-Galactosidase activity was measured after growth in various media such as LB with or without glucose, minimal medium containing glycerol, sorbitol, maltose or glucose. β-Galactosidase activity is measured in Miller units (ONPG units converted to OD ₆₀₀ per ml/min).

In fig. 6 shows the level of lacZ expression from a single-copy expression cassette integrated into the genome containing either the glpF or glpT promoter sequence (SEQ ID NO: 54 or SEQ ID NO: 50), a 54-nucleotide fragment of glpF-5'UTR (SEQ ID NO: 36) and SEQ ID NO: 10 or the following variants:

(A) Variants SEQ ID NO: 10;

(B) measurements of β-galactosidase activity (the reporter gene is expressed from constructs containing 10 variants of SEQ ID NO: 10 that have a modified RBS. β-galactosidase activity is measured in Miller units (ONPG units converted to OD ₆₀₀ per ml/min) .

In fig. Figure 7 shows the effect on lacZ expression (measured as the level of β-galactosidase activity) after modification of the -10 region of the glpF promoter. Seven different constructs containing the glpF promoter (SEQ ID NO: 12) and its variants (PglpF_19, PglpF_20, PglpF_17, PglpF_11, PglpF_13 or PglpF_9) were functionally linked to lacZ and inserted (in a single copy) into the E. coli genome, and activity of the reporter gene, lacZ, was assessed as the level of β-galactosidase activity measured in Miller units (ONPG units converted to OD600 per ml/min). All designs contain SEQ ID NO: 10.

In fig. Figure 8 shows the effect of shortening the 5' end of the glpF sequence on the expression of the lacZ gene. Truncated variants of the glpF promoter (SEQ ID NO: 54) (the promoter sequence was truncated by 15, 140, 165 or 180 base pairs from the 5' end) are functionally linked to lacZ and are expressed from a single copy integrated into the E. coli genome. All constructs contain the 54 nucleotide fragment glpF-5'UTR (SEQ ID NO: 36) and SEQ ID NO: 10. β-Galactosidase activity is determined in Miller units (ONPG units converted to OD ₆₀₀ per ml/min).

In fig. Figure 9 shows the effect on the level of β-galactosidase activity after disruption of the glpR gene in a host cell from a construct containing lacZ operably linked to the glpF promoter (SEQ ID NO: 54, linked to a 54-nucleotide fragment of glpF-5. UTR (SEQ ID NO : 36) and SEQ ID NO: 10. β-Galactosidase activity is measured in cells expressing the transcriptional repressor protein, GlpR (i.e., containing the native glpR gene), and in cells where the gene has been disrupted by introduction of kanR (i.e. into cells with low or no GlpR expression.) β-Galactosidase activity is determined in Miller units (ONPG units converted to OD ₆₀₀ per ml/min).

In fig. 10 shows the results of expression of heterologous genes in E. coli expressed from DNA constructs either under the lac promoter (Plac; gray circles) or the glpF promoter (PglpF; black circles) inserted as single copies into the E. coli genome. All constructs contain SEQ ID NO: 10 and native 5 ^r UTR fragments without RBS from the corresponding genes.

(A) 6′-sialyllactose (6′SL) production assessed in crude extracts of cells expressing α-2,6-sialyltransferase Pd2 (from Photobacterium damselae JT0160).

(B) 3′-Siallyllactose (3′SL) production assessed in crude extracts of cells expressing the α-2,3-sialyltransferase NST (from Neisseria meningitides MC58).

Sialyltransferase activity is measured as production in mM/h. In fig. 11 shows the levels of expression of lacZ under the control of the lac, glpF, glpA or glpT promoters from a multicopy plasmid containing one copy of the corresponding expression cassette. Each of the expression cassettes contains SEQ ID NO: 10 and native 5'UTR fragments without RBS from the corresponding genes (ie glpF, glpA, glpT or lacZ, respectively). β-Galactosidase activity is measured after growth in various media, such as LB with or without glucose (shaded or filled bars, respectively) in Miller units (ONPG units converted to OD ₆₀₀ per ml/min).

In fig. 12 shows the results of LNnT production in recombinant E. coli expressing heterologous galT and lgtA genes under the control of either the Plac or PglpF promoter (both expression cassettes contain SEQ ID NO: 10 and native 5 ^r UTR fragments without RBS from lacZ or glpF, respectively ).

(A) MDO1 expresses galT and lgtA with Plac in a high- and medium-copy number plasmid, respectively. MP1497 expresses lgtA from one chromosomal copy of the gene using PglpF and galT in a high copy number plasmid using Plac. MP1499 expresses galT from a single copy of the chromosomal gene using PglpF and lgtA in a mid-copy number plasmid using Plac.

(B) MP2622 and MP166 express lgtA and galT from one or three chromosomally integrated gene copies, respectively, using Plac; MP1825 expresses lgtA and lgtA from single chromosomal copies using PglpF.

- 4 044676

In fig. 13 shows the results of LNT production in recombinant E. coli expressing heterologous galTK and lgtA genes under the control of either Plac or PglpF (both expression cassettes containing SEQ ID NO: 10 and RBS-less native 5 ^r UTR fragments from lacZ or glpF, respectively).

(A) MDO15 expresses galTK and lgtA under the control of Plac in high- and medium-copy number plasmids, respectively. MP1498 expresses lgtA with one chromosomally integrated copy of the gene using PglpF and galTK in a high copy plasmid using Plac. MP1655 expresses galTK from two chromosomal copies of the gene using PglpF, and lgtA in a mid-copy number plasmid using Plac.

(B) MP245 expresses 3 and 2 chromosomally integrated copies of the lgtA and galTK genes, respectively, using Plac; MP1920 expresses lgtA and galTK from single chromosomally integrated gene copies using PglpF.

In fig. 14 shows the results of LNFP-I production in recombinant E. coli expressing heterologous galTK, lgtA and futC genes under the control of PglpF. MP2239 and MP2374 express lgtA, galTK, and futC from single chromosomally integrated gene copies using PglpF. In addition, MP2374 contains an additional copy of the colanic acid genes gmd, wcaJ (fcI), wcaH (gmm), wcaI, cpsB (manC), and cpsG (manB), which are all expressed under the control of PglpF. The expression cassettes contain SEQ ID NO: 10 and a native RBS 5 ^r -less UTR fragment from glpF.

In fig. 15 shows the results of 3'SL production in recombinant E. coli expressing heterologous nst, neuA, neuB and neuC genes. MAP425 expresses 2 chromosomally integrated copies of nst as well as neuA, neuB and neuC from a high copy number plasmid using Plac. MAP1214 expresses nst, neuA, neuB, and neuC from a single chromosomal copy of the gene using PglpF. Expression cassettes contain SEQ ID NO: 10 and native RBS 5 ^r -less UTR fragments from lacZ or glpF, respectively.

In fig. 16 shows the results of 6'SL production in modified E. coli expressing the heterologous genes Pd2, neuA, neuB and neuC. MAP265 expresses one chromosomal copy of Pd2, as well as neuA, neuB, and neuC from a high-copy plasmid using Plac. MAP1200 expresses Pd2, neuA, neuB, and neuC from a single chromosomal copy of the gene using PglpF. Expression cassettes contain SEQ ID NO: 10 and native RBS 5 ^r -less UTR fragments from lacZ or glpF, respectively.

In fig. Figure 17 shows the results of 2'FL production in recombinant E. coli expressing the heterologous futC gene: strain FT18 contains two plasmids expressing futC and the colanic acid genes gmd, fcl, manC and manB under the control of Plac, strain MAP965 contains a single copy of futC and genes colanic acid: gmd, wcaJ (fcI), wcaH (gmm), wcaI, cpsB (manC) and cpsG (manB), which are expressed under the control of PglpF. Expression cassettes contain SEQ ID NO: 10 and native 5 ^r UTR fragments lacking RBS from lacZ or glpF, respectively.

Detailed Description of the Invention

The present invention generally relates to DNA constructs and expression systems suitable for the recombinant production of biological molecules. In particular, the present invention relates to recombinant bacterial expression systems capable of providing stable and exceptionally high expression of a gene that is operably linked to a promoter and a synthetic non-coding DNA sequence located upstream of the gene, wherein said synthetic DNA sequence (interchangeably referred to herein as synthetic/artificial/recombinant DNA sequence (i)), contains a fragment of the 5' untranslated leader DNA sequence (5'UTR DNA) of the glp gene from Escherichia coli (E. coli) and the DNA sequence CAAGGAGGAAACAGCT (SEQ ID NO: 10) or its variant. The sequence CAAGGAGGAAACAGCT (SEQ ID NO: 10) is an artificial DNA sequence that was originally derived from the 5'UTR of lacZ from E. coli and was modified into a ribosome binding site (RBS) sequence. The sequence was previously described for its ability to enhance expression of a reporter gene (lacZ) by approximately 6-fold in a model gene expression system using a nucleotide construct, where the sequence was operably linked to an artificial promoter and to a 30-nucleotide DNA sequence capable of stabilizing mRNA. (Meynial-Salles I. et al. (2005), Appl. Eviron. Microbiol., 71:2140-2144; WO 03/089605). The inventors unexpectedly discovered that SEQ ID NO: 10 or a variant thereof, when not linked to a described RNA stabilizing DNA sequence, is not able to enhance expression of a reporter gene (lacZ) from all randomly selected promoters, but only from a few of them. However, if SEQ ID NO: 10 is associated with a fragment of the 5'UTR DNA of the glp gene, predominantly the glpF, glpA, glpT or glpD gene, the expression of the reporter gene is significantly increased and the level of gene expression is independent or much less dependent on the choice of promoter, i.e. e. promoter strength has less effect on the level of gene expression when the synthetic DNA sequence of the invention is inserted between the promoter and the gene.

Accordingly, the first aspect of the invention relates to a synthetic non-coding DNA sequence (i) comprising a first DNA fragment and a second DNA fragment, wherein the first fragment

- 5 044676 is a 5'UTR-,HHK fragment of a glp gene, preferably a glpF, glpA, glpT or glpD gene, and the second fragment is SEQ ID NO: 10 or a variant thereof, and wherein the second fragment is located below the first fragment (i.e. i.e. the second fragment is connected to the end of the first fragment). Preferably, the synthetic non-coding DNA sequence (i) is part of a nucleotide construct where it is operably linked to a promoter DNA sequence (ii) and optionally to a coding DNA sequence (iii) and wherein said synthetic DNA sequence is located downstream of the promoter DNA sequence (ii) and optionally upstream of the coding DNA sequence (iii). The term does not necessarily mean in the present context that, in some embodiments, the invention relates to nucleotide constructs that include a promoter DNA sequence (ii) and a synthetic DNA sequence (i), but not coding DNA (iii). In other embodiments, the synthetic DNA sequence (i) may be operably linked to the coding DNA sequence (iii) and the promoter DNA is not included in the construct. Still, in other embodiments, the construct may comprise a synthetic DNA sequence (i) and contain neither a promoter DNA nor a coding DNA sequence. Nucleotide constructs containing a synthetic DNA sequence (i) can be inserted into the host cell genome upstream of the gene and downstream of the gene's native promoter, for example, replacing an existing native DNA sequence, or it can be inserted into the genomic DNA to replace either or both from the promoter and/or 5'UTR DNA sequences of the genomic gene of interest. The construct of the invention can also be used to modify the expression of a gene of interest in a desired manner (ie, to increase or decrease gene expression) relative to natural gene expression controlled by native (not artificially modified) genomic DNA regulatory sequences. As indicated, in one embodiment, the construct may contain only the artificial DNA sequence (i), i.e. have neither a promoter nor a coding DNA sequence, since such a construct can be inserted into the genome of the host cell below the genomic promoter sequence and above the gene/coding sequence (replacing an existing native sequence or adding/extending an existing sequence), and due to This changes the expression of the gene (increases or decreases) compared to its natural expression from the corresponding genomic promoter. In other embodiments, the construct of the invention may comprise an operably linked promoter DNA sequence (ii), a synthetic DNA sequence (i), and a coding DNA sequence (iii) that encodes a heterologous or homologous (with respect to the host cell) biological molecule, where the synthetic DNA sequence is located between the promoter DNA and the coding DNA sequences.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention relates. Singleton et al. (1994), Dictionary of Microbiology and Molecular Biology, second edition, John Wiley and Sons (New York) will provide one skilled in the art with a general vocabulary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described. Most of the nomenclature and general laboratory procedures required in this application can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (2012); Wilson K. and Walker J., Principles and Techniques of Biochemistry and Molecular Biology (2010), Cambridge University Press; or Maniatise et al., Molecular Cloning A laboratory Manual, Cold Spring Harbor Laboratory (2012); or in Ausubel et al., Current protocols in molecular biology, John Wiley and Sohns (2010). Hereafter, the guidelines are referred to as Sambrook et al., Wilson & Walker, Maniatise et al., Ausubel et al. respectively.

Unless otherwise specified, the terms defined in the specification apply to all aspects and embodiments of the invention. All embodiments described in the specification and working examples apply to all any aspects of the invention.

As used herein, the term nucleic acid includes RNA, DNA, and DNA molecules. It is understood that, as a result of the degeneracy of the genetic code, a variety of nucleotide sequences encoding a given protein can be obtained. The term nucleic acid is used interchangeably with the term polynucleotide. An oligonucleotide is a short-chain nucleic acid molecule. A primer is an oligonucleotide, occurring naturally, for example in a purified restriction product or produced synthetically, that is capable of acting as a starting point for synthesis when exposed to conditions that produce a primer extension product that is complementary to the nucleic acid strand (i.e. e. in the presence of nucleotides and an inducing agent such as DNA polymerase, and at a suitable temperature and pH). The primer is preferably single-stranded for maximum amplification efficiency, but alternatively can be double-stranded. If the primer is

- 6 044676 Since it is double-stranded, it is first processed to separate the chains before being used to produce chain extension products. Preferably, the primer is a deoxyribonucleotide. The primer must be long enough to allow synthesis of extension products in the presence of an inducing agent. The exact length of the primers will depend on many factors, including temperature, primer source, and method application.

Nucleic acid construct means an artificially engineered segment of nucleic acid, particularly a segment of DNA, that is intended to be transplanted into a target cell, such as a bacterial cell, to modify the expression of a genomic gene, or to express a gene/coding DNA sequence that may be included in the construct. In the context of the invention, the nucleic acid construct comprises a recombinant DNA sequence essentially consisting of optionally one, two or three distinct DNA sequences: a synthetic non-coding DNA sequence (i) containing a ribosome binding site (RBS), a promoter DNA sequence (ii) and a coding sequence DNA (iii). In embodiments involving a construct comprising two or three of the listed sequences, the sequences in the construct are operably linked to each other. Functionally linked is defined herein as a configuration in which a regulatory sequence, i.e. the promoter sequence and/or the 5UTR sequence are suitably positioned relative to the coding DNA sequence such that the regulatory sequences direct transcription of the coding sequence and translation of the mRNA into the polypeptide sequence encoded by the coding DNA. In an embodiment where the construct comprises a coding DNA sequence, it is preferred that the coding DNA encodes at least one protein or RNA molecule that has an activity that is directly or indirectly involved in the production of one or more HMOs in a host cell (i.e. activity is essential or beneficial for the production of one or more HMOs). Non-limiting examples of such activities include: enzymatic activity, gene expression regulating activity, chaperone activity. Non-limiting examples of coding DNA sequences (iii) are described below and in the working examples. The DNA construct of the invention is, in some embodiments, referred to as an expression cassette of the invention. The DNA constructs/expression cassettes of the invention, in some embodiments, may contain more than one DNA coding sequence, which may encode different biological molecules. Preferably, the constructs (containing one or more DNA coding sequences (iii)) contain one copy of the promoter DNA sequence (ii) and one copy of the synthetic DNA sequence (i). The DNA constructs of the present invention can be inserted into a plasmid DNA/vector, transferred into a target/host cell, and expressed from plasmids and/or chromosomes. DNA constructs can be linear or circular. A linear or circular DNA construct inserted into a bacterial genome or expression plasmid is interchangeably referred to herein as an expression cassette, expression cartridge, or cartridge. In one embodiment, the cartridge is a linear DNA construct containing three DNA sequences: a promoter (DNA sequence (ii)), a synthetic DNA sequence (i)) downstream of the promoter, and a coding DNA sequence (sequence (iii)) encoding a biological molecule of interest. The construct may also contain additional sequences, such as a transcription terminator sequence and two terminal flanking regions that are homologous to the genomic region and that allow homologous recombination, and/or other sequences described herein. The cartridge can be produced by methods well known in the art, for example, using standard methods described in Wilson & Walker. The use of a linear expression cartridge may provide the advantage that the genomic insertion site can be freely selected by appropriate selection of flanking homologous regions of the cartridge. Thus, insertion of a linear expression cartridge allows for greater variability per region of the genome. Linear cartridges are included in preferred embodiments of the invention.

The term ribosome binding site (RBS) refers to a short nucleotide sequence, typically of about 4-16 nucleobases, that functions by positioning the ribosome on the mRNA molecule for translation of the encoded protein. A modified ribosome binding site is a ribosome binding site in which one or more base pairs have been changed. According to the invention, the synthetic non-coding DNA sequence (i) contains RBS in SEQ ID NO: 10 or a variant thereof. Variants of SEQ ID NO: 12 may be used in another embodiment, for example, to modify the expression of a genomic DNA sequence of interest, as, for example, we show herein that some variants may enhance gene expression to an even greater extent than achieved expression using SEQ ID NO: 10, while other variants may reduce gene expression. Useful but non-limiting embodiments of placenta

- 7 044676 DNA bodies that include RBS can be found in the table below. 1 and described in the specification.

For purposes of this invention, a promoter or promoter region or promoter element is a nucleotide sequence that is recognized and bound by DNA-dependent RNA polymerase during transcription initiation. A promoter, together with other transcriptional and translational regulatory nucleotide sequences (also called regulatory sequences), is required for the expression of a given gene or group of genes (operon). In general, transcriptional and translational control sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. The transcription start site refers to the first nucleotide to be transcribed and is designated +1. Nucleotides downstream of the start site are numbered +2, +3, +4, etc., and nucleotides in the opposite 5' direction (up) are numbered -1, -2, -3, etc. The promoter of the invention is a single DNA sequence. The term single DNA sequence means that the sequences are not an integrated fragment of genomic DNA, but an artificial/cloned DNA fragment that is identical or homologous to the genomic DNA sequence; Following this definition, the promoter DNA of the construct/expression cassette described herein is considered to be derived from the genomic DNA sequence contained in the promoter region of the gene. The promoter DNA sequence of the construct of the invention may be derived from the promoter region of any gene from the genome of the selected species, preferably from the promoter region of the genomic DNA of E. coli. According to the invention, any promoter DNA sequence that is capable of binding to RNA polymerase and initiating transcription is suitable for practicing the invention. As already indicated, the nucleotide sequence of the promoter DNA construct may be identical or have a certain percentage identity, such as about 65-70% identity, preferably at least 80% identity, preferably from about 90% to about 99.9% identity with the nucleotide the sequence of a fragment of a genomic DNA sequence, preferably a bacterial genomic DNA sequence, which is considered to be the promoter region of a single gene or operon, for example the glp or lac operons of E. coli. The terms about, around and approximately are used interchangeably and mean a deviation from a specified value of 1-10% or a slight deviation that does not affect the corresponding characteristic. An operon is a functional unit of genomic DNA containing a cluster of genes under the control of a single promoter. The glp operon refers to a group of genes involved in the respiratory metabolism of glycerol in bacteria. The invention in preferred embodiments relates to four E. coli glp operons, in particular glpFKX, glpABC, glpTQ and glpD. The promoters of these operons are referred to herein as the glpF, glpA, glpT and glpD promoters and are abbreviated herein as PglpF, PglpA, PglpT and PglpD, respectively. In some other embodiments, the invention provides an E. coli lac operon comprising the Z, Y and A genes and their lac promoter (abbreviated herein as Plac). It is preferred that the promoter sequence of the glp operon contained in the promoter DNA construct of the invention be identical or have at least 80% identity, preferably 90-99.9% identity, with the nucleotide sequence of the E. coli genomic DNA fragment located before the sequences having identifiers (ID) Genetic Bank: EG10396 (glpF), EG10391 (glpA), EG10394 (glpD), EG10401 (glpT), EG10527 (lacZ). The E. coli genome herein refers to the complete genomic DNA sequence of E. coli K-12 MG1655 (GenBank ID: U00096.3). Selected, but not limited to, embodiments of the promoter DNA sequences of the invention can be found in the table below. 1 and described in the application.

The promoter DNA sequence (ii) may contain several structural features/elements such as regulatory regions capable of influencing (facilitating or inhibiting) RNA polymerase binding in the cell and initiating transcription of the downstream (3' direction) coding sequence such as, for example, binding sites for transcriptional regulatory proteins, such as, for example, the transcriptional repressor protein GlpR, or the transcriptional activator protein CRP. The regulatory region includes protein-binding domains (consensus sequences) responsible for binding RNA polymerase, such as box -35 and box -10 (Pribnow box). All of the mentioned regulatory sequences of the promoter DNA construct may have a certain percentage of identity with the corresponding genomic promoter sequences, i.e. the invention provides the original DNA sequences (native/wild type) or variants thereof. Some non-limiting useful embodiments of promoter DNA sequence variants (ii) can be found in the table below. 1 and described in the specification.

The promoter sequence of the invention preferably contains at least 50 nucleotides, more preferably at least 60 nucleotides, such as about 65 to about 100 nucleotides, about 75 to about 115 nucleotides, about 85 to about

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125 nucleotides, for example from 90 to 115, 110-120, 120-130, 130-140, 140-150 or more than 150 nucleotides, for example 155-165, 165-175, 175-185, 185-195, 195-205, 205-215, 215-225, 225-235, 235-245 245-255, 255-265, 250-350. In some embodiment, the promoter sequence may be up to 500-1000 nucleotides in length. In some embodiments, the selected promoter sequence may also be shorter, i.e. include less than 50 nucleotides. The length of the promoter DNA sequence is not a general limiting factor of the invention, since the invention in another embodiment provides for any promoter DNA sequence (i) that is capable of binding to RNA polymerase and initiating transcription of a gene in an expression cassette or a gene of interest in the genome. In one preferred embodiment, the promoter DNA is derived from the genomic promoter region of the glp operon, for example, the glpF promoter region (PglpF), the glpA promoter region (PglpA), the glpT promoter region (PglpT), the glpD promoter region (PglpD). In some preferred embodiments, the promoter DNA sequence (i) may be selected from any of the DNA sequences set forth herein as SEQ ID NO: 48, 49, or 54; in some embodiments, a variant of said sequence may be preferred. Non-limiting useful examples of such embodiments are described herein and are shown in Table 1. 1. In one preferred embodiment, the promoter DNA sequence has its origin from E. coli genomic DNA (GenBank ID EG10396), in particular from the region of genomic DNA containing the promoter element of the glpFXK operon (in particular the 50-350 nucleotide DNA sequence upstream transcription of the glpFXK operon or a variant thereof that has at least 90% sequence identity). One preferred embodiment of the promoter DNA sequence (i) is SEQ ID NO: 54.

In various embodiments, the invention may relate to a promoter that is inducible/regulated or continuously active. The term inducible promoter means that the activity of the promoter, i.e. the ability of a promoter to initiate and maintain transcription of a functionally related gene at a certain level is regulated by an external factor, such as a carbon source molecule. In some embodiments, the activity of a promoter of the invention can be controlled by the presence or absence of a carbon source molecule in the medium, such as glycerol, glucose, arabinose, etc. A carbon source generally refers to a carbohydrate that can be taken up and metabolized by the bacterial cell. Preferably, the inducible promoter of the invention is a carbon source-inducible E. coli glp promoter, preferably a glpF, glpA, glpD, glpT promoter, or a variant thereof that is inducible by the same carbon source as the corresponding parent promoter. Preferably, the promoter contains at least one cyclic AMP receptor protein (cAMP-CRP) binding site. In other embodiments, the invention relates to a promoter that is not inducible, i.e. to promoter activity that is independent of induction, for example, by a carbon source. Preferably, the latter promoter is a glp promoter of the invention, the DNA structure of which has been modified to make the promoter activity carbon source independent or to make the promoter constitutively active, for example by removing/modifying GlpR binding sites in the promoter sequence.

As stated above, the invention also relates to variant promoter DNA sequences that can be used in the constructs of the invention. By variant in the present context is meant an artificial nucleic acid sequence that preferably has about 70-99% similarity to the nucleotide sequence of the promoter DNA sequence in question. The percentage of similarity of compared nucleic acid sequences indicates the proportion of sequences that have an identical structure, i.e. identical nucleotide composition. The percentage of sequence similarity for purposes of the invention can be determined using any method well known in the art, such as BLAST. The scope of the term variant includes nucleotide sequences complementary to the DNA sequences described herein, mRNA sequences and synthetic nucleotide sequences, for example, PCR primers, and other oligonucleotides that are related to the nucleotide sequences of the constructs of the invention.

According to the invention, the construct/expression cassette contains a synthetic non-coding DNA sequence (i) which contains a ribosome binding site (RBS). The term synthetic DNA sequence in the present context means a man-made DNA sequence, i.e. The DNA sequence is artificially constructed and consists of at least two DNA fragments, wherein at least one of the fragments is derived from genomic DNA (i.e., it corresponds to the DNA sequence of genomic DNA), and the other DNA fragment is an artificial DNA sequence , containing about 16 nucleic bases, which preferably does not correspond to the sequence of natural bacterial genomic DNA containing RBS. Specifically, the synthetic DNA sequence (i) consists of two DNA fragments: a first DNA fragment and a second DNA fragment. The first DNA fragment is approximately 70-100% identical

- 9 044676 sequence identity with a fragment of a genomic DNA sequence derived from an untranslated DNA sequence located downstream of the transcription initiation point and upstream of the start of translation of a gene, for example in the glp gene. Preferably, the first fragment contains a chain of at least 5 to 80 contiguous nucleotides, for example 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-30 nucleotides, 30-40 nucleotides, 40-50 nucleotides, 50 -60 nucleotides after the transcription start point (starting at nucleotide +2) of the glp gene, preferably the glpF, glpA or glpD gene. The DNA sequence of the first fragment may be homologous or heterologous to the DNA promoter (ii) and/or the coding DNA sequence (iii). Homologous in this context means that the DNA sequence of the first fragment is derived from the 5'UTR region of a gene that is naturally located downstream of the promoter of the construct, or is a naturally occurring fragment of the 5'UTR of the gene of the construct, or is in nature associated with both; heterologous means that the first DNA fragment does not correspond to a fragment of the natural (genomic) 5'UTR region associated with the gene or promoter construct. In one preferred embodiment, the first DNA fragment has its origin from the genomic DNA sequence of the 5'UTR of the glp gene, preferably the glpF gene. Preferably, it is heterologous to the coding DNA sequence (iii). In some embodiments, it may be preferred that the DNA sequence of the first DNA fragment be homologous to the DNA sequence of the promoter (ii); in other embodiments, it may be preferable that said first sequence be heterologous to the DNA sequence of promoter (i). In one preferred embodiment, the first DNA fragment is or contains the nucleotide sequence set forth in SEQ ID NO: 36.

The second artificial DNA sequence fragment (i) is the nucleotide sequence CAAGGAGGAAACAGCT (SEQ ID NO: 10) or a variant thereof. Some useful non-limiting embodiments of SEQ ID NO: 10 are the nucleotide sequences of SEQ ID NO: 38-47. In some embodiments of the construct of the invention, the first DNA fragment (DNA sequence 5'-UTR) is located downstream of the promoter DNA sequence (DNA sequence (ii)) (and upstream of the second DNA fragment); and the second DNA fragment is located upstream of the coding DNA sequence (iii) (i.e., before the translation initiation point), i.e. in an artificial DNA sequence (i) the first DNA fragment precedes the second DNA fragment. One preferred embodiment of the synthetic DNA sequence (i) is SEQ ID NO: 37.

Non-limiting DNA sequences (i) and (ii), fragments, variants and combinations thereof useful in various aspects of the invention are described in table. 1.

Table 1

Name·. SEQ ID: Sequence (5’->3’) Description PglpF 5'U TR-glpFd!6nb SEQ GO NO: 1 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA A 284-nucleotide DNA fragment obtained from the genomic DNA of E. soy (ref. sequence.

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GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTACAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA C ID U00096.3), located at 16 nucleotides upstream of the codon initiation of translation of the glpF gene PglpA 5'U TR-glpAdl6nb SEQ ID NO: 2 GAAAACATTCATAAATTAAATGTGAATT GCCGCACACATTATTAAATAAGATTTAC AAAATGTTCAAAATGACGCATGAAATCA CGTTTCACTTTCGAATTATGAGCGAATA TGCGCGAAATCAAACAATTCATGTTTTTT ACTATGGCTAAATGGTAAAAAAACGAA 166- nucleotide fragment of DNA obtained from the genomic DNA of E. soy (ref. sequence. ID U00096.3), located at 16 nucleotides upstream of the codon initiation of translation of the glpA gene PglpD 5’U TR-glpDdl6 nb SEQ ID NO: 3 TGCGTCTCTCTTTCTTTACAAACAAGTGG GCAAATTTACCGCACAGTTTACGTCGAA GCGGCAGATAAACGCCATAATGTTATAC ATATCACTCTAAAATGTTTTTTCAATGTT ACCTAAAGCGCGATTCTTTGCTAATATG TTCGATAACGAACATTTATGAGCTTTAA CGAA 174- nucleotide fragment of DNA obtained from genomic DNA of E. coli (ref. sequence. ID U00096.3), located at 16 nucleotides upstream of the codon initiation of gene translation

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glpD PglpT- 5'UTRglpT-dl6nb SEQ ID NO: 4 CCATTTAGCCATAGTAAAAAAACATGAATT GTTTGATTTCGCGCATATTCGCTCATAAT TCGAAAGTGAAACGTGATTTCATGCGTC ATTTTGAACATTTTGTAAATCTTATTTAA TAATGTGTGCGGCAATTCACATTTAATTT ATGAATGTTTTCTTAACATCGCGGCAAC TCAAGAAACGGCAGGTTCTCTCACTGAA TCAGGCTGTTAATCATAAATAAGACCAC GG A 229-nucleotide DNA fragment obtained from the genomic DNA of E. soy (ref. sequence ID U00096.3), located at 16 nucleotides upstream of the codon initiation of translation of the glpT gene 16nb -glpF SEQ ID NO: 5 TCTTCAGGATCCGATT A 16-nucleotide DNA fragment obtained from E. coli genomic DNA (ref. sequence ID U00096.3), located immediately upstream of the codon initiation of translation of the glpF gene 16nb-glpA SEQ ID NO: 6 CTTCAGAGGGATAACA A 16-nucleotide DNA fragment obtained from the genomic DNA of E. coli (ref. sequence ID U00096.3), located directly

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upstream codon initiation glpA gene translation 16nb-glpD SEQ ID NO: 7 AGTGAATGAGGGCAGC A 16-nucleotide DNA fragment obtained from E. coli genomic DNA (ref. sequence ID U00096.3), located upstream of the codon initiation of translation of the glpD gene 16nb -glpT SEQ ID NO: 8 GCCACGGAGGCTATCA A 16-nucleotide DNA fragment obtained from E. coli genomic DNA (ref. sequence ID U00096.3), located immediately upstream of the codon initiation of translation of the glpT gene 16bnb-lacZ SEQ ID NO: 9 CACACAGGAAACAGCT A 16-nucleotide DNA fragment obtained from E. coli genomic DNA (ref. sequence ID U00096.3), located upstream of lacZ

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mutl6bp lacZ (recRBS) SEQ ID NO: 10 CAAGGAGGAAACAGCT Option SEQ ID NO: 9) (CAC -> AGG) Plac_org SEQ ID NO: 11 TGTGAGTTAGCTCACTCATTAGGCACCC CAGGCTTTACACTTTATGCTTCCGGCTCG TATGTTGTGTGGAATTGTGAGCGGATAA CAATTTCACACAGGAAACAGCT 107 nucleotide DNA fragment located upstream of lacZ, obtained from genomic DNA of E. soy (ref. sequence ID U00096.3); 1asoperon promoter element PglpF_54n b 5 'URTglpF_recRB S (PglpF_rec ) SEQ ID NO: 12 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTA CAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA CCAAGGAGGAAACAGCT 300 nucleotide DNA fragment containing SEQ Ш NO: 1 and SEQ ID NO: 10 PglpF_SDl SEQ ID NO:13 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTACAAGCATCGTGGAGGTCCG 300 nucleotide DNA fragment containing SEQ ID NO: 1 and SEQ ID NO: 38 (see figure 6)

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TGACTTTCACGCATACAACAAACATTAA CCAAATTCGAAACAGCT PglpF_SD2 SEQ ID NO: 14 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTA CAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA CCAAGCGCAAAACAGCT 300 nucleotide DNA fragment containing SEQ ID NO: 1 and SEQ ID NO: 39 (see figure 6) PglpF_SD3 SEQ ID NO:15 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTA CAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA CCAAGAACAAAACAGCT 300 nucleotide DNA fragment containing SEQ ID NO: 1 and SEQ Ш NO: 40 (see figure 6) PglpF_SD4 SEQ ID NO:16 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTA CAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA CCAACTAGGAAACAGCT 300 nucleotide DNA fragment containing SEQ Yu NO: 1 and SEQ GO NO:41 (see figure 6)

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PglpF_SD5 SEQ ID NO: 17 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTACAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA CCAACCGAGAAACAGCT 300 nucleotide DNA fragment containing SEQ ID NO: 1 and SEQ ID NO: 42 (see figure 6) PglpF_SD6 SEQ ID NO:18 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTA CAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA CCAAGAGCTAAACAGCT 300 nucleotide DNA fragment containing SEQ ID NO: 1 and SEQ ID NO:43 (see figure 6) PglpF_SD7 SEQ ID NO:19 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTA CAAGCATCGTGGAGGTCCG TGACTTTCAGCATACAACAAACATTAA CCAAGAGCAAAACAGCT 300 nucleotide DNA fragment containing SEQ ID NO: 1 and SEQ Ш NO:44 (see Figure 7)4 PglpF_SD8 SEQ ID NO:20 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTTCTCCTGGTGACAT 300- nucleotide

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AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTACAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA CCAAGAAAAACAG C.T. DNA fragment containing SEQ ID NO: 1 and SEQ ID NO:45 (see figure 7) PglpF_SD9 SEQ ID NO:21 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTA CAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA CCAAAGGAAAAACAGCT 300 nucleotide DNA fragment containing SEQ ID NO: 1 and SEQ Ш NO:46 (see figure 7) PglpF_SDl 0 SEQ ID NO:22 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTA CAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA CCAACTGAGAAACAGCT 300 nucleotide DNA fragment containing SEQ I NO: 1 and SEQ W NO: 47 (see figure 7) PglpF_9 SEQ ID NO:23 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA Variant SEQ ID NO: 12 containing modification

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TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATTTAATTACA GCATGCCTACAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA CCAAGGAGGAAACAGCT area -10 (see figure 7) PglpF_l 1 SEQ ID NO:24 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATCAGAATAC AGCATGCCTACAAGCATCGTGGAGGTCC GTGACTTTCACGCATACAACAAACATTA ACCAAGGAGAAACAGCT Variant SEQ ID NO: 12 containing modification of region -10 (see figure 7) PglpF_13 SEQ ID NO:25 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATATCCTTCCT ACAGCATGCCTACAAGCATCGTGGAGGT CCGTGACTTTCACGCATACAACAAACAT TAACCAAGGAGGAAACAGCT Variant SEQ ID NO: 12 containing modification of region -10 (see figure 7) PglpF_17 SEQ ID NO:26 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA Variant SEQ ID NO: 12 containing modification of region -10 (see figure 7)

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GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAATGATAC AGCATGCCTACAAGCATCGTGGAGGTCC GTGACTTTCACGCATACAACAAACATTA ACCAAGGAGAAACAGCT PglpF_19 SEQ ID NO:27 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATGAAGCTAC AGCATGCCTACAAGCATCGTGGAGGTCC GTGACTTTCACGCATACAACAAACATTA ACCAAGGAGAAACAGCT Variant SEQ ID NO: 12 containing modification of region -10 (see figure 7) PglpF_20 SEQ ID NO:28 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATCAGTATACA GCATGCC TACAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA CCAAGGAGGAAACAGCT Variant SEQ ID NO: 12 containing modification of region -10 (see figure 7) D15PglpF SEQ ID NO:29 GATTACGGTTTGCCACACTTTTCATCCTT CTCCTGGTGACATAATCCACATCAATCG AAAATGTTAATAAATTTGTTGCGCGAAT GATCTAACAAACATGCATCATGTACAAT CAGATGGAATAAATGGCGCGATAACGCT CATTTTATGACGAGGCACACACATTTTA AGTTCGATATTTCTCGTTTTTGCTCGTTA ACGATAAGTTTACAGCATGCCTACAAGC 285 nucleotide DNA fragment of SEQ ID NO: 12

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ATCGTGGAGGTCCGTGACTTTCACGCAT ACAACAAACATTAACCAAGGAGGAAAC AGCT D140PglpF SEQ ID NO:30 ATGGCGCGATAACGCTCATTTTATGACG AGGCACACACATTTTAAGTTCGATATTT CTCGTTTTTGCTCGTTAACGATAAGTTTA CAGCATGCCTACAAGCATCGTGGAGGTC CGTGACTTTCACGCATACAACAAACATT AACCAAGGAGGAAACAGCT 160- well cleotid DNA fragment of SEQ ID NO: 12 D165PglpF SEQ ID NO:31 ACGAGGCACACACATTTTAAGTTCGATA TTTCTCGTTTTTGCTCGTTAACGATAAGT TTACAGCATGC CTACAAGCATCGTGGAGGTCCGTGACTT TCACGCATACAACAAACATTAACCAAGG AGGAAACAGCT 135- well cleotid DNA fragment of SEQ ID NO: 12 D180PglpF SEQ ID NO:32 TTTAAGTTCGATATTTCTCGTTTTTTGCTC GTTAACGATAAGTTTACAGCATGCCTAC AAGCATCG TGGAGGTCCGTGACTTTCACGCATACAA CAAACATTAACCAAGGAGGAAACAGCT 120- well cleotid DNA fragment of SEQ ID NO: 12 PglpA_- 5'UTRglpA_recRB S (rec PglpA) SEQ ID NO:33 GAAAACATTCATAAATTAAATGTGAATT GCCGCACACATTATTAAATAAGATTTAC AAAATGTTCAAAATGACGCATGAAATCA CGTTTCACTTTCGAATTATGAGCGAATA TGCGCGAAATCAAACAATTCATGTTTTTT ACTATGGCTAAATGGTAAAAACGAAC AAGGAGGAAACAGCT 182- well cleotid DNA fragment comprising SEQ ID NO: 2 and SEQ ID NO: 10 PglpD 5'U TRglpD_recR B.S. (rec PglpD) SEQ ID NO:34 TGCGTCTCTCTTTCTTTACAAACAAGTGG GCAAATTTACCGCACAGTTTACGTCGAA GCGGCAGATAAACGCCATAATGTTATAC ATATCACTCTAAAATGTTTTTTCAATGTT ACCTAAAGCGCGATTCTTTGCTAATATG TTCGATAACGAACATTTATGAGCTTTAA CGAACAAGGAGGAAACAGCT 190- well cleotid DNA fragment containing SEQ ID NO: 3 and SEQ ID NO: 10 PglpT 5'U SEQ ID CCATTTAGCCATAGTAAAAAAACATGAATT 245-

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TRglpT_recRB S (rec PglpT NO:35 GTTTGATTTCGCGCATATTCGCTCATAAT TCGAAAGTGAAACGTGATTTCATGCGTC ATTTTGAACATTTTGTAAATCTTATTTAA TAATGTGTGCGGCAATTCACATTTAATTT ATGAATGTTTTCTTAACATCGCGGCAAC TCAAGAAACGGCAGGTTCTCTCACTGAA TCAGGCTGTTAATCATAAATAAGACCAC GGCAAGGAGGAAACAGCT nucleotide fragment of DNA containing SEQ ID NO: 4 and SEQ ID NO: 10 54nb 5'UTRglpF SEQ ID NO:36 TGCCTACAAGCATCGTGGAGGTCCGTGA CTTTCACGCATACAACAAACATTAAC A 54-nucleotide fragment of DNA about the 5'UTR-glpF located downstream of the site initiation transcriptions and 16 nucleotides upstream codon initiation broadcasts synDNA(i) (70UTR) SEQ ID NO:37 TGCCTACAAGCATCGTGGAGGTCCGTGA CTTTCACGCATACAACAAACATTAACCA AGGAGGAAACAGCT 70-nucleotide synthetic non-coding sequence of DNA (ii) containing SEQ ID NO: 36 and SEQ ID NO: 10 recRBS_vl (SD1) SEQ ID NO:38 CAAATTCGAAACAGCT Option SEQ ID NO: 10 (see Fig. 6, A) recRBS_v2 (SD2) SEQ ID NO:39 CAAGCGCAAAACAGCT Option SEQ ID NO: 10 (see Fig. 6, A) recRBS_v3 SEQ ID CAAGAACAAAACAGCT Option SEQ ID

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(SD3) NO:40 NO: 10 (see Fig.6, A) recRBS_v4 (SD4) SEQ ID N0:41 CAACTAGGAAAACAGCT Option SEQ ID NO: 10 (see Fig. 6, A) recRBS_v5 (SD5) SEQ ID NO:42 CAACCGAGAAACAGCT Option SEQ ID NO: 10 (see Fig. 6, A) recRBS_v6 (SD6) SEQ ID NO:43 CAAGAGCTAAACAGCT Option SEQ ID NO: 10 (see Fig. 6, A) recRBS_v7 (SD7) SEQ ID NO:44 CAAGAGCAAAACAGCT Option SEQ ID NO: 10 (see Fig. 6, A) recRBS_v8 (SD8) SEQ ID NO:45 CAAGAGAAAAACAGCT Option SEQ ID NO: 10 (see Fig. 6, A) recRBS_v9 (SD9) SEQ ID NO:46 CAAAGGAAAAACAGCT Option SEQ ID NO: 10 (see Fig. 6, A) recRBS_vl 0 (SD10) SEQ ID NO:47 CAACTGAGAAACAGCT Option SEQ ID NO: 10 (see Fig. 6, A) PglpA_org SEQ ID NO:48 GAAAACATTCATAAATTAAATGTGAATT GCCGCACACATTATTAAATAAGATTTAC AAAATGTTCAAAATGACGCATGAAATCA CGTTTCACTTTCGAATTATGAGCGAATA TGCGCGA 119- nucleotide fragment of DNA obtained from the genomic DNA of E. soy (ref. sequence. ID U00096.3), located above the point transcription initiation

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glpA; element glpA promoter PglpT_org SEQ ID NO:49 CCATTTAGCCATAGTAAAAAAACATGAATT GTTTGATTTCGCGCATATTCGCTCATAAT TCGAAAGTGAAACGTGATTTCATGCGTC ATTTTGAACATTTTGTAAATCTTATTTAA TAATGTGTGCGGCAATTCACATTTAATTT ATGAATGTTTTCTTAACATCGCGGGCA 169- nucleotide fragment of DNA obtained from genomic DNA of E. coli (ref. sequence. ID U00096.3), located above the point glpT transcription initiation; element glpT promoter PglpA_70U TR SEQ ID NO:50 GAAAACATTCATAAATTAAATGTGAATT GCCGCACACATTATTAAATAAGATTTAC AAAATGTTCAAAATGACGCATGAAATCA CGTTTCACTTTCGAATTATGAGCGAATA TGCGCGATGCCTACAAGCATCGTGGAGG TCCGTGACTTTCACGCATACAACAAACA TTAACCAAGGAGGAAACAGCT 189 nucleotide DNA fragment obtained by combining SEQ ID NO: 49 with SEQ ID NO 37 PglpT_70U TR SEQ ID NO: 51 CCATTTAGCCATAGTAAAAAAACATGAATT GTTTGATTTCGCGCATATTCGCTCATAAT TCGAAAGTGAAACGTGATTTCATGCGTC ATTTTGAACATTTTGTAAATCTTATTTAA TAATGTGTGCGGCAATTCACATTTAATTT ATGAATGTTTTCTTAACATCGCGGGCA TGCCTACAAGCATCGTGGAGGTCCGTGA CTTTCACGCATACAACAAACATTAACCA AGGAGGAAACAGCT 239 nucleotide DNA fragment obtained by combining SEQ ID NO: 50 with synthetic non-coding sequence DNA(s) (SEQ ID NO 37)

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PglpT_70U TR_SD4 SEQ ID NO:52 CCATTTAGCCATAGTAAAAAAACATGAATT GTTTGATTTCGCGCATATTCGCTCATAAT TCGAAAGTGAAACGTGATTTCATGCGTC ATTTTGAACATTTTGTAAATCTTATTTAA TAATGTGTGCGGCAATTCACATTTAATTT ATGAATGTTTTCTTAACATCGCGGGCA TGCCTACAAGCATCGTGGAGGTCCGTGA CTTTCACGCATACAACAAACATTAACCA ACTAGGAAACAGCT 239 nucleotide DNA fragment SEQ GO NO: 50 where 16 π.o. located above the site translation initiations, identical to SEQ ID NO 41 PglpT_70U TR_SD9 SEQ ID NO:53 CCATTTAGCCATAGTAAAAAAACATGAATT GTTTGATTTCGCGCATATTCGCTCATAAT TCGAAAGTGAAACGTGATTTCATGCGTC ATTTTGAACATTTTGTAAATCTTATTTAA TAATGTGTGCGGCAATTCACATTTAATTT ATGAATGTTTTCTTAACATCGCGGGCA TGCCTACAAGCATCGTGGAGGTCCGTGA CTTTCACGCATACAACAAACATTAACCA AAGGAAAAACAGCT 239 nucleotide DNA fragment SEQ GO NO: 50, where 16 bp, located above the site translation initiations, identical to SEQ ID NO 46 PglpF_org SEQ ID NO:54 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCA 230 nucleotide DNA sequence, obtained from E. coli genomic DNA (ref. sequence ID U00096.3), located above the point glpF transcription initiation; element glpF promoter

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PglpT- 5 'UTRglpT_org SEQ ID NO:55 CCATTTAGCCATAGTAAAAAAACATGAATT GTTTGATTTCGCGCATATTCGCTCATAAT TCGAAAGTGAAACGTGATTTCATGCGTC ATTTTGAACATTTTGTAAATCTTATTTAA TAATGTGTGCGGCAATTCACATTTAATTT ATGAATGTTTTCTTAACATCGCGGCAAC TCAAGAAACGGCAGGTTCTCTCACTGAA TCAGGCTGTTAATCATAAATAAGACCAC GGGCCACGGAGGCTATCA 245 nucleotide DNA sequence, obtained from the genomic DNA of E. soy (ref. sequence ID U00096.3), located upstream of the site glpT translation initiation PglpA- 5 'UTRglpA_org SEQ ID NO:56 GAAAACATTCATAAATTAAATGTGAATT GCCGCACACATTATTAAATAAGATTTAC AAAATGTTCAAAATGACGCATGAAATCA CGTTTCACTTTCGAATTATGAGCGAATA TGCGCGAAATCAAACAATTCATGTTTTTT ACTATGGCTAAATGGTAAAAAAACGAA CTTCAGAGGGATAACA 182 nucleotide DNA sequence, obtained from E. coli genomic DNA (ref. sequence ID U00096.3), located upstream of the site glpA translation initiation PglpF 5'UTRglpF_org SEQ ID NO:57 GCGGCACGCCTTGCAGATTACGGTTTGC CACACTTTTCATCCTTTCTCCTGGTGACAT AATCCACATCAATCGAAAATGTTAATAA ATTTGTTGCGCGAATGATCTAACAAACA TGCATCATGTACAATCAGATGGAATAAA TGGCGCGATAACGCTCATTTTATGACGA GGCACACACATTTTAAGTTCGATATTTCT CGTTTTTGCTCGTTAACGATAAGTTTACA GCATGCCTACAAGCATCGTGGAGGTCCG TGACTTTCACGCATACAACAAACATTAA CTCTTCAGGATCCGATT 300 nucleotide DNA sequence, obtained from E. coli genomic DNA (ref. sequence ID U00096.3), located upstream of the site glpF translation initiation PglpD 5'UTRglpD_org SEQ ID NO: 10 4 TGCGTCTCTCTTTCTTTACAAACAAGTGG GCAAATTTACCGCACAGTTTACGTCGAA GCGGCAGATAAACGCCATAATGTTATAC ATATCACTCTAAAATGTTTTTTCAATGTT ACCTAAAGCGCGATTCTTTGCTAATATG TTCGATAACGAACATTTATGAGCTTTAA CGAA AGTGAATGAGGGCAGC 190 nucleotide DNA sequence, obtained from E. coli genomic DNA (ref. sequence ID U00096.3), located above the point glpD translation initiation

As stated above, in some preferred embodiments, the construct includes three operably linked DNA sequences: a premotor DNA sequence(s), a synthetic non-coding DNA sequence (i), and at least one coding DNA sequence (iii) (gene). The coding DNA sequence (iii) is an isolated DNA sequence that is approximately 70-100% identical to the sequence of a fragment of genomic DNA containing a gene encoding a biological molecule, such as a protein or RNA. The coding DNA (iii) of the construct may be homologous or heterologous to the promoter

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DNA sequences (ii). Heterologous in the present context means that the expression of the corresponding genomic coding DNA sequence is typically controlled by a promoter other than the promoter of the construct. Accordingly, homologous in the present context means that the corresponding genomic sequences of the promoter DNA sequence (ii) and the coding DNA sequence (iii) are naturally related in the genome of the parent species.

By the term coding nucleotide sequence is meant a nucleotide sequence that contains a set of consecutive non-overlapping triplets (codons) that are transcribed into mRNA and translated into a polypeptide when under the control of appropriate control sequences, i.e. promoter The boundaries of the coding sequence are usually defined by the ribosome binding site located immediately upstream of the open reading frame at the 5' end of the mRNA, the transcription start codon (AUG, GUG, or UUG), and the translation stop codon (UAA, UGA, or UAG). The coding sequence may include, but is not limited to, genomic DNA, cDNA, synthetic and recombinant nucleotide sequences.

In a preferred embodiment, the coding nucleotide sequence of the construct of the invention is heterologous with respect to the promoter and, in some embodiments, also with respect to the first DNA fragment of non-coding DNA sequence (i) in the construct. However, with respect to the host cell in which the coding DNA is to be expressed, said DNA may be either heterologous (ie, derived from another species or genus) or homologous (ie, derived from a host cell). For example, in one embodiment, the DNA coding sequence of the construct may encode a biological molecule, such as a protein, that is foreign to the host, i.e. the nucleotide sequence of the coding DNA is heterologous with respect to the host species because it is of origin from a donor species that is different from the host organism, or the nucleotide sequence of the coding DNA contains a modification that results in the expression of a polypeptide that is not identical to the polypeptide expressed with the corresponding unmodified Host DNA sequences, i.e. an artificially modified DNA coding sequence originally derived from a host is considered heterologous in the present context. In the case where the host is a particular prokaryotic species, the heterologous nucleotide sequence may have its origin in another genus family, another order or class, another phylum (division), or another domain (superkingdom) of organisms. A heterologous nucleotide sequence derived from a donor other than the host may be modified before its introduction into a host cell by mutations, insertions, deletions or substitutions of individual nucleic acids or part of the heterologous nucleotide sequence as long as such modified sequences have the same function ( functionally equivalent) to the reference sequence. Heterologous nucleotide sequence as defined herein also includes nucleotide sequences originating from another domain (superkingdom) of organisms such as eukaryotes (eukaryotic origin), such as, for example, enzymes involved in the synthesis or degradation of human milk oligosaccharides (HMOs). . However, in other embodiments, the encoding nucleic acid may be homologous to the host cell. The term homologous nucleotide sequence (used synonymously herein as a nucleotide sequence native to a host or a nucleotide sequence derived from a host) as used herein means that the nucleotide sequence is derived from (or derived from) the same organism, or the same genus family, or the same order or class, the same phylum (division), or the same domain (superkingdom) of organisms as the host organism. In one embodiment, the coding DNA of a construct described herein may encode an enzyme or sugar transport protein that is typically expressed by a bacterial host cell that naturally contains in its genome genes encoding said enzyme or sugar transport protein.

In general, the invention contemplates any coding DNA, since any coding DNA can be included in a construct of the invention and transcribed from a promoter included in the construct. In some preferred embodiments, the coding DNA encodes a protein, such as an enzyme, a transport protein, a regulatory protein, a chaperone, etc. The term protein is referred to interchangeably herein as a polypeptide. In other preferred embodiments, the coding DNA may encode a regulatory (non-coding) RNA molecule (ncRNA), such as, for example, functionally important types of non-coding RNAs such as transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), as well as small RNAs such as microRNAs , miRNAs and long ncRNAs. In a preferred embodiment, at least one DNA coding construct of the invention encodes a protein or RNA associated with the synthesis, degradation or transport of human milk oligosaccharides, precursors or derivatives thereof. At least one coding DNA sequence means that the construct in different embodiments may contain more than one coding DNA sequence, for example two coding sequences

- 26 044676 sti, such as the first and second coding sequence; three coding sequences, such as the first, second and third coding sequences, etc. Preferably, the multiple coding DNA sequences in these embodiments are expressed in tandem and transcription is controlled by a single copy of the promoter DNA (ii) construct. First, second, third, etc. The coding DNA sequences may, in various embodiments, encode various enzymes or other proteins whose function is essential or beneficial for HMO production by the host cell, such as enzymes, transporter proteins, regulatory proteins, chaperones, etc. By essential in this context is meant that the protein is directly involved in the synthesis of HMO, for example, it is an enzyme that assists in the process of producing HMO from a precursor of HMO, for example an enzyme with glucosyltransferase activity. By useful in this context is meant that the protein is not directly involved in the synthesis of HMO, but it contributes to a process that is beneficial for the production of HMO by the host cell, for example, a protein that facilitates the transport (into or out of the host cell) of HMO or a HMO precursor. Some non-limiting protein variants that are considered herein to be essential for the production of one or more HMOs by a host cell can be found in Table. 2, and proteins that are considered useful for the production of one or more HMOs by a host cell are listed in table. 3 below.

table 2

Gene Sequence, ID (GenBank) Description Example of CME IgtA WP_002248149.1 β-1,3-Ν- acetylglucosaminyltransferase LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFPV, LNFP-VI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH galT NP_207619.1 β-1,4- galactosyltransferase LNnT, LNFP-III, LNFP-VI, pLNH, F-pLNH I, pLNnH cpsIBJ AB050723 β-1,3- galactosyltransferase LNT, LNFP-I, LNFP-II, LNFP-V, LNDFH-I, LNDFH-II, pLNH, F-pLNH I MAMA_R 764 AGC02224.1 α-1,3-fucosyltransferase 2’FL, 3’FL, DFL, LNFP-I, LNFP-III, LNFP-V, LNFPVI, LNDFH-II, F-pLNH I Mg791 AEQ33441.1 α-1,3-fucosyltransferase 2’FL, 3’FL, DFL, LNFP-I, LNFP-III, LNFP-V, LNFPVI, LNDFH-II, F-pLNH I Moumou_ 00703 AGC02224.1 α-1,3-fucosyltransferase 2’FL, 3’FL, DFL, LNFP-I, LNFP-III, LNFP-V, LNFPVI, LNDFH-II, F-pLNH I futA NP_207177.1 α-1,3-fucosyltransferase 2’FL, 3’FL, DFL, LNFP-I, LNFP-III, LNFP-V, LNFPVI, LNDFH-II, F-pLNH I futC CP003904 α-1,2-fucose yltransferase 2'FL, DFL, LNFP-I, LNDFH-I fucT AAB81031.1 α-1,3-fucosyltransferase 2’FL, 3’FL, DFL, LNFP-I, LNFP-III, LNFP-V, LNFPVI, LNDFH-II, F-pLNH I

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fucTIII AY450598 a-1,4-fucose yltransferase LNDFH-I, LNDFH-II fucTa AF194963 a-1,3/4- fucosyltransferase LNFP-II, LNDFH-I, LNDFH-II Pd2.6ST BAA25316.1 a-2,6-sialyltransferase 6'SL PspST6 BAF92026.1 a-2,6-sialyltransferase 6'SL PiST6_14 5 BAF91416.1 a-2,6-sialyltransferase 6'SL PiST6_ll 9 BAI49484.1 a-2,6-sialyltransferase 6'SL NST AAC44541.1 a-2,3-sialyltransferase 3'SL neuA AF400048 CMP-Neu5 Ac synthetase 3'SL, 6'SL neuB AF400048 Sialic acid synthase 3’SL, 6’SL, sialic acid neuC AF400048 O1cIAc-6-phosphate-2epimerase 3’SL, 6’SL, sialic acid

Table 3

Gene Sequence, ID (UniProt) Description CME products, description gmd POAC88 OOP-mannose-4,6- dehydratase 2’FL, 3’FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFHI, LNDFH-II, FpLNHI wcaG P32055 GDP fucose synthase 2’FL, 3’FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFHI, LNDFH-II, FpLNHI wcaH P32056 GDP-mannose- mannosyl hydrolase 2’FL, 3’FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFHI, LNDFH-II, FpLNHI cpsB P24174 Mannose-1-phosphate guanylyltransferase 2'FL, 3'FL, DFL, LNFP-I, LNFP-II,

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LNFP-III, LNFP-V, LNFP-VI, LNDFHI, LNDFH-II, FpLNH I cpsG P24175 Phosphomannomutase 2’FL, 3’FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFHI, LNDFH-II, FpLNHI glmS P17169 E-glutamine-O-fructose-6phosphate aminotransferase LNT, LNnT, LNFPI, LNFP-II, LNFPIII, LNFP-V, LNFPVI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH, 3’SL, 6’SL glmU P0ACC7 Merged N- acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase LNT, LNnT, LNFPI, LNFP-II, LNFPIII, LNFP-V, LNFPVI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH, 3’SL, 6’SL glmM P3112O Phosphoglucosamine mutase LNT, LNnT, LNFPI, LNFP-II, LNFPIII, LNFP-V, LNFPVI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH, 3’SL, 6’SL ampG P0AE16 Muropeptide: H+symporter LNT, LNnT, LNFP- I, LNFP-II, LNFP- III, LNFP-V, LNFP- VI, LNDFH-I,

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nagA P0AF18 N-acetylglucosamine bphosphate deacetylase LNDFH-II, pLNH, F-pLNH I, pLNnH, 3’SL, 6’SL LNT, LNnT, LNFPI, LNFP-II, LNFPIII, LNFP-V, LNFP- VI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH, 3’SL, 6’SL N-acetyl-O- LNT, LNnT, LNFP- glucosamine kinase I, LNFP-II, LNFP- III, LNFP-V, LNFP- nagK P75959 β-N-acetyl hexosaminid aza VI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH, 3’SL, 6’SL LNT, LNnT, LNFPI, LNFP-II, LNFPIII, LNFP-V, LNFP- nagZ P75949 DNA-binding VI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH, 3’SL, 6’SL LNT, LNnT, LNFP- transcription double I, LNFP-II, LNFP- PhoP regulator III, LNFP-V, LNFP- phop P23836 VI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH, 3’SL, 6’SL Glutamine synthetase LNT, LNnT, LNFP- glnA P0A9C5 I, LNFP-II, LNFP- III, LNFP-V, LNFP- VI, LNDFH-I,

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LNDFH-II, pLNH, F-pLNH I, pLNnH, 3’SL, 6’SL ppk P0A7B1 Polyphosphate kinase LNT, LNnT, LNFPI, LNFP-II, LNFPIII, LNFP-V, LNFPVI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH, 3’SL, 6’SL pykA P21599 pyruvate kinase II LNT, LNnT, LNFPI, LNFP-II, LNFPIII, LNFP-V, LNFPVI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH, 3’SL, 6’SL pgm P36938 Phosphoglucomutase LNT, LNnT, LNFPI, LNFP-II, LNFPIII, LNFP-V, LNFP- VI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH galU P0AEP3 UTP - glucose-1-phosphaturidyl transferase LNT, LNnT, LNFPI, LNFP-II, LNFPIII, LNFP-V, LNFP- VI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH galE P09147 iBP-glucose-4-epimerase LNT, LNnT, LNFPI, LNFP-II, LNFPIII, LNFP-V, LNFPVI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH

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DNA-binding transcriptional dual regulator NagC LNT, LNnT, LNFP- I, LNFP-II, LNFP- III, LNFP-V, LNFP- nagC P0AF20 Glucokinase VI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH, 3’SL, 6’SL 2'FL, 3'FL, DFL, LNFP-I, LNFP-II, LNFP-ΙΠ, LNFP-V, glK P0A6V8 6-phosphofructokinase II LNFP-VI, LNDFH- I, LNDFH-II, FpLNHI 2'FL, 3'FL, DFL, LNFP-I, LNFP-II, LNFP-ΙΠ, LNFP-V, pfkB P06999 Xanthine-guanine phosphoribosyltransferase LNFP-VI, LNDFH- I, LNDFH-II, FpLNHI 2'FL, 3'FL, DFL, LNFP-I, LNFP-II, LNFP-ΙΠ, LNFP-V, gpt P0A9M5 G wanylate kinase LNFP-VI, LNDFH- I, LNDFH-II, FpLNHI 2'FL, 3'FL, DFL, LNFP-I, LNFP-II, LNFP-ΙΠ, LNFP-V, gmk P60546 Nucleoside diphosphate kinase LNFP-VI, LNDFH- I, LNDFH-II, FpLNHI 2'FL, 3'FL, DFL, LNFP-I, LNFP-II, ndk P0A763 NADP+-dependent glucose 6-phosphate dehydrogenase LNFP-ΙΠ, LNFP-V, LNFP-VI, LNDFHI, LNDFH-II, FpLNHI 2'FL, 3'FL, DFL, LNFP-I, LNFP-II, LNFP-ΙΠ, LNFP-V, zwf P0AC53 iTP: glucose-1-phosphate uridylyltransferase LNFP-VI, LNDFH- I, LNDFH-II, FpLNHI LNT, LNnT, LNFP- I, LNFP-II, LNFP- III, LNFP-V, LNFP- galF P0AAB6 VI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH

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The term human milk oligosaccharide or HMO in this context refers to the complex carbohydrate found in human breast milk (see Urashima et al., Milk Oligosaccharides. Nova Science Publisher (2011); or Chen, Adv. Carbohydr. Chem. Biochem., 72, 113 (2015)). HMOs have a basic structure containing a lactose unit at the reducing end, which can be extended by one or more e-N-acetyllactosaminyl and/or one or more β-lacto-N-biosyl units, and this basic structure can be replaced by a-L-fucopyranosyl and/or a-N-acetylneuraminyl (sialyl) group. In this regard, non-acidic (or neutral) HMOs lack a sialyl residue, while acidic HMOs have at least one sialyl residue in their structure. Non-acidic (or neutral) HMOs can be fucosylated or non-fucosylated. Examples of such neutral non-fucosylated HMOs include lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH) and lacto-N-hexaose (LNH). Examples of neutral fucosylated HMOs include 2'fucosyllactose (2'-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3-FL), difucosyllactose ( DFL), lacto-N-fucopentase II (LNFP-II), lacto-N-fucopentaose III (LNFP-II I), lacto-N-difucohexaose III (LNDFH-III), fucosyl-lacto-N-hexaose II (FLNHII), lacto-N-fucopentase V (LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-para-lacto-N-hexaose I (FpLNH -I), fucosyl-p-lacto-N-neohexaose II (F-pLNnH II) and fucosyl-lacto-N-neohexaose (FLNnH). Examples of acidic HMOs include 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl-3'-sialyllactose (FSL), 3'-O-sialyllacto-N-tetraose ( LST a), fucosyl-LST a (FLST a), 6'-O-sialyllacto-N-tetraose b (LST b), fucosyl-LST b (FLST b), b'-O-sialyllacto-Nneotetraose (LST c) , fucosyl-LST c (FLST c), 3'-O-sialyllacto-N-neotetraose (LST d), fucosyl-LST d (FLST d), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N -neohexaose I (SLNH-I), sialyl-lacto-Nneohexaose II (SLNH-II) and disialyl-lacto-14-tetraose (DSLNT). In the context of the present invention, lactose is considered as a representative of HMO.

The term HMO precursor in the present context refers to a compound involved in the biosynthetic pathway of one or more HMOs of the invention that are produced and naturally present in the host cell or imported into the cell from the extracellular environment. Some non-limiting examples of CME precursors are listed below.

Predecessor Product UDP-GlcNAc LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, 3'SL, 6'SL, pLNnH, ( F)LSTa, (F)LSTb, (F)LSTc, (F)LSTd UDP-Gal LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, pLNH, F-pLNH I, pLNnH, LSTa, LSTb, LSTc, LSTd GDP-fucose LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, F-pLNH I, 2'FL, 3'FL, DFL, FLSTa, FLSTb , FLSTc, FLSTd

The term HMO transporter means a biological molecule, such as a protein, that facilitates the transport/export of HMO synthesized by a host cell across a cell membrane, for example, into the cellular environment, or the transport/import of HMO from the cellular environment into the cellular cytosol.

The term HMO derivative means a molecule that is derived from an HMO molecule or contains an HMO group, for example a ganglioside molecule, an artificial carbohydrate/protein structure containing an HMO group.

The expression cassette of the present invention can be used for the recombinant production of one or more HMOs, either genomically integrated or as part of a plasmid, or, in some embodiments, the host cell can contain both a genomically integrated and plasmidized expression cassette, wherein at least at least one or both expression cassettes contain one or more genes that are necessary and/or useful for the production of one or more HMOs, and wherein the expression of at least one of these genes is under the control of a glp promoter according to the invention (i.e. PglpF, PglpA , PglpD or PglpT, preferably PglpF). Preferably, the genome-integrated cassette contains at least one (or a first set) of DNA coding sequences, and the plasmid cassette contains at least one second coding DNA (or a second set of DNA coding sequences), wherein at least one first and/or at least one second DNA coding sequences are operably linked to the glp promoter of the invention. In some preferred embodiments, at least one of the expression cassettes is expressed under the control of Pglp, for example, the coding sequence of the genome-integrated cassette is operably linked to a glp promoter of the invention, for example PglpF, and the plasmid coding sequence is operably linked to another promoter, for example lac- promoter, or another promoter. In some embodiments,

- 33 044676 both genome-integrated and plasmid cassettes can be expressed under the control of the same or a different glp promoter of the invention, for example, the genome-integrated cassette promoter is PglpF and the plasmid promoter is PglpA. In other embodiments, all expression cassettes contained in a host cell may contain the same glp promoter. In one preferred embodiment, the host cell contains at least one copy of a genome-integrated expression cassette of the invention containing PglpF. Preferably, the host cell genome contains one or a small number of copies of the genome-integrated expression cassette, for example two or three copies. However, in some embodiments, the host may contain multiple copies of the expression plasmid, where each plasmid contains one copy of the expression cassette of the invention. In some embodiments, the host cell may contain several different nucleotide constructs of the invention, both genome-integrated and/or plasmid-based. Each of several different nucleotide constructs may be inserted into the genome of a host cell or into a plasmid in a single copy or in multiple copies. In some embodiments, it is preferable for the constructs to be embedded in a single copy or a low number of copies.

According to the invention, one copy of the expression cassette of the invention, contained in a host cell, either integrated into the genome or in a plasmid, can provide the number of biological molecules encoded by the DNA coding sequence (ii) (preferably under the control of a glp promoter, e.g. PglpF) , which is sufficient to ensure a high level of production of one or more HMOs by the host cell. Surprisingly, a single genome-integrated copy of the expression cassette of the present invention can provide levels of HMO production that are comparable to or higher (eg, 2-10 times higher) than the production levels achieved using expression from the same cassette in a high-copy plasmid ( 100-500 copies). In some embodiments, it may be advantageous to express two or more genes associated with HMO production in a host cell. Genes associated with NMO can be included in a single construct and expressed in tandem from one (or multiple) copies in the genome or from a plasmid; or the genes may be included in various constructs of the invention, and one gene is expressed from a cassette integrated into the genome and the other gene from a plasmid. In other embodiments, a different expression method, composition, or copy number of the expression cassettes may be provided. Preferably, at least one gene included in the above expression cassettes encodes a protein with enzymatic activity that is necessary for the synthesis of HMO in the host cell. Non-limiting gene variants that may be preferentially expressed under the control of the glp promoter are described in Table 1. 2 and 3 and in working examples.

In accordance with the above, the second aspect of the invention relates to a recombinant cell containing a nucleotide construct according to the invention. A recombinant cell is referred to interchangeably herein as a host cell. Preferably the host cell is a bacterial cell. The terms bacterial host species and bacterial host cell are used interchangeably to refer to a bacterial cell that has been transformed to contain a DNA construct of the invention and is capable of expressing a heterologous polypeptide encoded by a corresponding heterologous DNA coding sequence of the construct. The terms transformation, transformed and transplanted are synonymous and refer to the process in which an extracellular nucleic acid, such as a vector containing a construct of the invention, with or without accompanying material, enters a host cell. Transformation of suitable host cells, for example, with an expression vector can be accomplished by well known methods such as electroporation, conjugation, or chemical methods such as calcium phosphate-mediated transformation and natural transformation systems described, for example, in Maniatis et al. or in Ausubel et al.

As far as bacterial host cells are concerned, there are in principle no restrictions; they can be eubacteria (Gram-positive or Gram-negative) or archaebacteria if they allow genetic manipulation to insert a gene of interest and can be grown on a commercial scale. Preferably, the host cell allows cultivation to high cell densities. Non-limiting examples of bacterial host cells that are suitable for the recombinant industrial production of the HMOs of the invention include Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum or Xanthomonas campestris. Bacteria of the genus Bacillus can also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus and Bacillus circulans. Also, bacteria of the genera Lactobacillus and Lactococcus can be modified using the methods of the present invention, including, but not limited to, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crisp atus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also

- 34 044676 suitable bacterial species for the invention described herein. Also included as part of this invention are strains modified as described herein from the genera Enterococcus (eg, Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (eg, Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp. ., Micrococcus spp., Rhodococcus spp., and Pseudomonas (eg, Pseudomonas fluorescens and Pseudomonas aeruginosa). Bacteria having the characteristics described herein are cultured in the presence of lactose, and the HMO produced by the cell is recovered either from the bacterium itself or from the culture supernatant of the bacteria. HMO is purified using a suitable procedure available in the art (for example, such as described in WO 2015188834, WO 2017182965 or WO 2017152918).

In a preferred embodiment, the host cell is E. coli. However, as already mentioned, various host cells can be used for the purposes of the invention.

One requirement for the host cell is that it contains a functional DNA-dependent RNA polymerase that can bind to the promoter and initiate transcription of the DNA construct. The RNA polymerase can be endogenous (native), homologous (recombinant), or foreign/heterologous (recombinant) to the host cell.

The construct of the invention, transformed into a selected bacterial host, can be expressed as a genome-integrated expression cassette or cloned into a suitable expression vector and expressed in a plasmid. In various embodiments, it may be preferable to use a genome-based expression system; in other embodiments, expression from a plasmid may be preferred. However, it is advantageous to use the construct of the invention in a genome-based expression system because, surprisingly, a single copy of the construct integrated into and expressed from the genome can provide high and stable levels of expression of the inserted gene product. An additional advantage is that genomic expression is stable over long periods of time. For the purposes of the invention, standard methods can be used for inserting the constructs of the invention into the genome of a host cell or into expression plasmids, which, for example, are described in Sambrook et al., Wilson & Walker, Maniatise et al. and Ausubel et al.

The terms transformation, transformed and transplanted are synonymous and refer to the process in which an extracellular nucleic acid, such as a vector containing a construct of the invention, with or without accompanying material, enters a host cell. Transformation of suitable host cells, for example with an expression vector, can be carried out by well known methods such as electroporation, conjugation, or chemical methods such as calcium phosphate-mediated transformation and natural transformation systems described, for example, in Maniatis et al. or in Ausubel et al.

For genome-based expression, there is a requirement for the host cell - the cell must be capable of homologous recombination (which is important for integrating the expression cartridge into the genome). Therefore, it is preferable that the host cell has a functional RecA recombination protein. However, since RecA can cause unwanted recombination events during culture, it is preferable that the host cell has a genomic mutation in its genomic recA site (rendering it dysfunctional) but instead has functional RecA provided by the recA sequence present on the helper plasmid. which can be removed (eliminated) after recombination using a thermosensitive replicon helper plasmid (Datsenko K.A. & Wanner B.L. (2000), Proc. Natl. Acad. Sci., USA, 97(12):6640-5). In view of recombination, in addition to RecA, it is preferable that the host cell contains DNA sequences encoding recombination proteins (eg, Exo, Beta and Gam). In this case, a host cell that already has this function can be selected, or a host cell can be created de novo by genetic engineering to insert these sequences.

With regard to the locus of integration, the expression system used in the invention allows for wide variability. In principle, any locus with a known sequence can be selected, provided that the sequence function is either optional or, if necessary, can be filled in (as in the case of auxotrophy, for example). Many integration loci suitable for the purposes of the invention are described in the prior art (see, for example, Francia VM & Lobo JMG (1996), J. Bacteriol., vl78, p. 894-898: Juhas M. et al. (2014), doi.org/10.1371/journal.pone.0111451; Juhas M & Aijoka FW (2015), Mcrobial. Biothechnol., v.8:617-748; Sabi A. et al. (2013), Mcrobial. Cell Factories, 12 :60).

The DNA construct can also be inserted in a site-specific manner. Considering site-specific gene insertion, another requirement for the host cell is that it contains at least one genomic region (either a coding or non-coding functional or non-functional region, or a region of unknown function) for which its sequence is known, and the structure of which can be disrupted or otherwise modified to allow insertion of a heterologous sequence without causing harm to the cell.

In some embodiments, the host cell carries in its genome a marker gene for

- 35 044676 conducting selection.

When choosing an integration locus, it must be taken into account that the frequency of DNA mutations caused by so-called adaptive evolution varies across the E. coli genome, and that the metabolic load triggered by chromosomally encoded expression of a recombinant gene can cause an increased frequency of mutations at the integration site. To obtain an expressing and stable host cell, it is preferable to select a highly conserved region of the genome as the integration site, which ensures a reduced mutation rate. Such highly conserved regions of the E. coli genome are, for example, genes encoding ribosomal components or genes involved in peptidoglycan biosynthesis, and these regions may preferably be selected for integration of the expression cartridge.

The genomic region of known sequence that can be selected for cartridge integration can be selected from the coding region of a non-essential gene or part thereof; from an optional non-coding functional region (i.e. promoter, transposon, etc.), from genes whose deletion may have beneficial effects in terms of production of a particular protein of interest, e.g. certain proteases, outer membrane proteins, potential product contaminants, genes encoding metabolic proteins (for example, important for the metabolism of sugar molecules that are undesirable or dispensable for a given host strain and/or fermentation process) or proteins for stress signaling pathways, for example, those that arise in response to harsh crop conditions, mechanism translational control of prokaryotes, which suppresses the synthesis of tRNA and rRNA during amino acid starvation. Alternatively, the integration site may be a marker gene that allows selection for the disappearance of said marker phenotype following integration. Alternatively, the site that can be selected for integration is a feature that, when removed, provides auxotrophy, i.e. the inability of an organism to synthesize a specific organic compound needed for its growth. In this case, the integration site may be an enzyme involved in biosynthesis or metabolic pathways; removal of such an enzyme results in an auxotrophic strain. Positive clones, i.e. those that carry an expression cassette may be selected for auxotrophy on the substrate or precursor molecules of said enzymes. Alternatively, the integration site may be an auxotrophic marker (non-functional, i.e., defective gene) that is replaced/supplemented by a corresponding prototrophic marker (i.e., a sequence that complements or replaces the defective sequence) present in the expression cassette, such thus allowing prototrophic selection.

In one aspect, the region is a nonessential gene. In one aspect, it may be a gene that itself is not essential to the cell. Essential bacterial genes are known from the literature, for example from the PEC (E. coli chromosome profiling) database (http://www.shigen.nig.ac.jp/ecoli/pec/genes.jsp) or from the so-called Keio Collection ( Baba et al., Molecular Systems Biology (2006), 2, 2006.0008). One example of a nonessential gene is RecA. Integration of the expression cassette at this site provides the genomic mutation described above in the context of the host cell requirements.

Suitable integration sites, eg sites that are easily accessible and/or are expected to provide higher levels of expression, can be identified in a preliminary screen. Such screening can be accomplished by generating a series of single deletion mutants according to the Keio collection (Baba et al., 2006), whereby the integration cartridge contains as variable elements different recombination sequences that have been preselected for specific insertion sites, and as constant elements, base sequences for integration and selection, including as a surrogate gene of interest a DNA sequence encoding a readily detectable protein under the control of an inducible promoter, for example, green fluorescent protein. The expression level of single knockout mutants thus generated can be easily quantified by fluorescence measurements. Based on the results of this procedure, a customized level of expression of the desired target protein can be achieved by changing the integration site and/or the number of inserted cartridges.

In embodiments in which the host cell contains DNA sequences encoding recombination proteins (e.g., Exo, Beta, and Gam as a feature of the parent cell or genetically engineered), integration may occur at the genomic site where these sequences are located recombination proteins. Upon integration of the expression cartridge, the sequences encoding the recombination proteins are destroyed or removed and therefore do not need to be removed in a separate step, as in the case of plasmid-encoded helper proteins.

Integration of a gene of interest into the bacterial genome can be achieved by conventional means, for example, using linear cartridges that contain flanking sequences homologous to a specific site in the chromosome, as described for the attTn7 site (Waddell C.S. and Craig N.L., Genes Dev. (1988), Feb, 2(2):137-49.); methods of genomic integration after

- 36 044676 sequences of nucleic acids in which recombination is mediated by the Red recombinase function of phage λ or the RecE/RecT recombinase function of prophage Rac (Murphy, J. Bacteriol. (1998), 180(8):2063-7; Zhang et al. , Nature Genetics (1998), 20:123-128; Muyrers et al., EMBO Rep. (2000), 1(3):239-243); in ways based on Red/ET recombination (Wenzel et al., Chem Biol. (2005), 12(3):349-56.; Vetcher et al., Appl Environ Microbiol. (2005), 71(4):1829 -35); or positive clones, i.e. clones that carry an expression cassette can be selected, for example, using a marker gene or a loss or gain of function gene.

In some embodiments, host cells are used that already contain a marker gene integrated into their genome, such as an antibiotic resistance gene or a gene encoding a fluorescent protein, such as GFP. In this case, an expression cartridge that does not contain the selectable marker is integrated into the chromosomal marker gene locus, and positive clones are selected for loss/disappearance of the corresponding phenotype, for example, they are selected for sensitivity to antibiotics or disappearance of fluorescence, which can be directly visualized on culture dishes . The advantage of these embodiments is that the marker is either disrupted or completely replaced by the expression cassette, and thus the functional marker sequence is not present after integration and does not require removal if this is not desired, as in the case of antibiotic resistance genes.

Alternatively, the marker gene is part of an expression cartridge. When the marker used for selection is a gene conferring resistance to antibiotics (eg kanamycin or chloramphenicol), positive clones are selected for antibiotic resistance (ie growth in the presence of the appropriate antibiotic). The marker gene (whether present in the host cell genome or introduced via an expression cartridge) can be removed upon integration of the cassette.

In some embodiments, the expression cell can be engineered to carry a defective selectable marker gene, such as an antibiotic resistance gene such as chloramphenicol or kanamycin, a fluorescent marker, or a gene involved in a sugar or amino acid metabolic pathway. In this case, the cartridge containing the gene of interest carries the missing part of the marker gene, and through integration, the marker gene regains its functionality. For example, the cartridge carries the missing portion of a marker gene at one of its ends and is integrated immediately next to the defective marker gene integrated into the genome, such that the fusion of the two fragments makes the marker gene complete and allows its functional expression. In the case of an antibiotic resistance gene, cells carrying an expression cassette are resistant to a particular antibiotic, in the case of a fluorescent marker, the cells can be visualized by fluorescence, and in the case of a metabolic pathway gene, the cells acquire the ability to metabolize the corresponding component. The advantage of this embodiment is that only a short portion of the marker gene cartridge needs to be synthesized, allowing the use of shorter or smaller insert cartridges compared to the prior art.

In some embodiments, selection of positive clones (ie, clones that carry an expression cassette) can be accomplished by correcting (ie, complementing) the auxotrophy of the host cell. In such embodiments, a host cell is used that has a mutation that has been selected for simple selection of positive transformed colonies, for example a strain that has a deletion or mutation that renders it unable to synthesize a compound that is required for its growth (such a mutation is called an auxotrophic marker) . For example, a bacterial mutant in which the proline synthesis pathway gene is inactivated is a proline auxotroph. Such a strain is unable to synthesize proline and can therefore only grow if proline can be extracted from the environment, unlike a proline prototroph which can grow in the absence of proline.

Any host cell having an auxotrophic marker can be used. It is preferable to use as auxotrophic markers mutations in genes necessary for the synthesis of amino acids, for example mutations in genes related to the synthesis of proline, leucine or threonine or co-factors such as thiamine. According to the invention, host cell auxotrophy is corrected by integrating the missing/defective gene into the genome as a component of an expression cartridge together with the integration of the gene of interest. The prototrophic cells thus obtained can be easily selected by growing them on so-called minimal medium (prototrophic selection), which does not contain the compound for which the original host cell is auxotrophic, allowing only positive clones to grow.

Prototrophic selection does not depend on the locus of integration. The locus of integration for prototrophic selection can be any gene in the genome or at a locus that carries an auxotrophic marker. A particular advantage of prototrophic selection is that after successful integration, neither an antibiotic resistance marker nor any other marker foreign to the host remains in the genome. Therefore, there is no need to remove these marker genes, which provides quick and easy

- 37 044676 simple procedure of cloning and selection. Another advantage is that restoring gene function is beneficial to the cell and provides greater system stability.

Alternatively, the marker gene that is inserted into the genome along with the expression cartridge may be a metabolic gene that is subject to a particular selection regime. Such a metabolic gene may allow a cell to grow on a specific (unusual) sugar or other carbon sources, and selection of positive clones can be achieved by growing cells on the specified sugar as the sole carbon source.

As described above, during long-term bacterial culture, adaptive evolution can cause an increased frequency of mutations at the integration site during expression of a chromosomally encoded recombinant protein. Using an auxotroph knockout mutant strain in combination with an expression cartridge that complements the missing function of the mutant strain (thereby generating a prototroph strain from an auxotroph mutant) has the added benefit that the restored gene provides benefits to the cell that give the cell a competitive advantage , so that cells in which adaptive evolution has occurred are suppressed. Thus, a method of negative selection for mutated clones is provided.

In some embodiments (where the protein of interest allows detection at the single cell or single colony level, such as by FACS or immunoassay (ELISA)), a marker gene is not required because positive clones can be identified by direct detection of the protein , of interest.

Integration methods to obtain an expression host cell are not limited to the integration of a single gene of interest at a single site in the genome; they allow for variability in both the integration site and the expression cassettes. For example, more than one gene of interest may be inserted, i.e. two or more identical or different sequences under the control of identical or different promoters can be integrated into one or more different loci in the genome. For example, it is possible to express two different proteins that form a heterodimeric complex. Heterodimeric proteins consist of two individually expressed protein subunits. One example of such a protein is an antibody molecule, such as the heavy and light chain of a monoclonal antibody or antibody fragment; other examples of heterodimeric proteins are CapZ, Ras, human helicase II, etc. These two monomer coding sequences may be present in a single expression cartridge that is inserted into a single integration locus. Alternatively, the two sequences may also be present in two different expression cassettes that are inserted independently of each other at two different integration loci. In any case, the promoters and induction modes may be the same or different.

Although the invention allows and can be advantageously used in practice for the plasmid-free production of biological molecules of interest encoded by a gene of a construct of the invention, it does not exclude that the expression system of the invention contains a plasmid that carries sequences to be expressed other than the gene of interest for example, helper proteins and/or recombination proteins described above. Naturally, care should be taken to ensure that in such embodiments the advantages of the invention are not impaired by the presence of the plasmid, i.e. Preferably, such a plasmid is present in low copy number and does not impose a metabolic burden on the cell.

The expression system used in the method according to the invention can be selected so that it contains virtually or completely no phage functions.

To summarize the above embodiments, the genomic expression of the expression cassettes of the invention provides the following major advantages:

With regard to the expression host construction procedure, the advantages are (i) a simple method for synthesizing and amplifying a linear insert cartridge; (ii) a high degree of flexibility (i.e., without restrictions) with respect to the locus of integration; (iii) high degree of flexibility regarding the selectable marker and the selection principle; (iv) the possibility of subsequent removal of the selectable marker; (v) a discrete and defined number of expression cartridges inserted (usually one or two).

Integration of one or more recombinant genes into the genome results in a discrete and predetermined number of genes of interest per cell. In the embodiment in which one copy of the gene is inserted, this number is typically one (unless the cell contains more than one genome, as occurs transiently during cell division), compared to plasmid-based expression, which is accompanied by an increase number of copies up to several hundred. In the expression system used in the method of the present invention, by freeing the host metabolism from plasmid replication, an increased proportion of the synthetic cellular machinery is used to produce the recombinant protein. Therefore, a design element can be used that allows strong expression of, for example, Pglp, such as PglpF, PglpA, PglpD or PglpT, without adverse effects on host metabolism by reducing copy content

- 38 044676 gene.

As stated above, plasmid-based expression systems have the disadvantage that during cell division, cells may lose the plasmid and therefore the gene of interest. This plasmid loss depends on several external factors and increases with the number of cell divisions (generations). This means that plasmid-based fermentations are limited in the number of generations (in conventional fermentations this number is approximately 20 to 50). In contrast, the genome-based expression system used in the method of the invention provides stable, predefined gene content for a virtually infinite number of generations and thus theoretically infinite culture time under controlled conditions (without the disadvantage of producing cells that do not produce the protein of interest , and with the only limitation of potentially occurring natural mutations, since they can occur in any gene).

In the case of chemically inducible promoters, the invention provides a particular advantage that the amount of inducer molecule when added, for example, in a continuous manner is directly proportional to the gene content per cell, either constant throughout the culture, or varies during the culture time in predetermined amounts. values. In this way, control of the rate of recombinant expression can be achieved, which is of great interest for regulating the rate of gene expression.

Because the genome-based expression system allows for precise control of protein expression, it is particularly advantageous in combination with signaling-targeted expression that depends on or relies on well-controlled expression.

As described above, the invention allows for simplified processes, improved process predictability and high reproducibility from fermentation to fermentation. The method of the invention using the expression system described above can be carried out in a fed-batch process, or in a semi-continuous or continuous mode, whereby the advantages of the genome-encoded expression system are optimally utilized. There are no restrictions on process parameters such as growth rate, temperature and culture medium components, except as determined by the requirements of the host cell and determined by the selected promoter.

Another advantage relates to the choice of inducer molecule: most of the available systems for high-level recombinant gene expression in E. coli are lac-based promoter-operator systems inducible by IPTG. The expression system used in the invention allows cultivation in a carbon-limited environment with continuous or pulsed supply of a carbon source, such as lactose, and provides tight control of the expression rate with a wide range of inexpensive carbon source inducers such as glycerol, fucose, lactose, glucose.

It is important that the expression system used in the invention has the advantage of providing a high yield of recombinantly produced biological molecules, both in terms of the concentration of the molecule per volume of culture medium (ie titer) and in terms of the content of the molecule in the resulting biomass. This feature makes the expression system used in the invention superior to prior art expression systems.

In addition, the invention offers the advantage that the selection of the host cell for expression and/or the optimal design of the expression cartridge can be easily achieved in preliminary screening tests. As an example, in such preliminary screens, a series of linear expression cartridges are designed that vary with respect to at least one element that affects the expression properties of the protein of interest (expression level or quality characteristics such as biological activity), namely regulatory elements (e.g., promoter and/or polymerase binding site) and/or sequence of the gene of interest (i.e., various codon usages), and/or recombination guide sequences, and/or any other elements in the cartridge, such as secretory leaders. The cartridge variants are inserted into the genome of a preselected host cell, and the resulting variant expression hosts are cultured, including induction of protein expression, under controlled conditions. By comparing protein expression, the host cell variant that shows the most favorable results from the point of view of the industrial production process is selected. In a variation of this preliminary approach, instead of identifying the optimal expression cartridge, the optimal bacterial strain can be identified by integrating identical cartridges into a panel of different host cells. Since the integration strategy has the advantage of allowing a discrete number of gene copies (eg, just one) to be integrated into the genome, prescreening for various parameters can be performed without interference from the plasmid replication process or changes in the plasmid copy number.

According to the invention, the term culture (or cultivation, also called fermentation) refers to the propagation of bacterial expressing cells in a controlled bio

- 39 044676 reactor in accordance with methods known in the industry.

The production of recombinant proteins is usually done through large-scale cultivation. The terms production and production scale within the meaning of the invention define fermentation with a minimum volume of 5 l of culture medium. Generally, a production scale process is defined as being capable of processing large volumes of a drug containing the recombinant protein of interest and producing quantities of the protein of interest that meet, for example in the case of a therapeutic protein, clinical trial requirements as well as for marketing. In addition to its large volume, the production scale method, as opposed to simple laboratory methods such as flask culture, is characterized by the use of a technical bioreactor (fermenter) system, which is equipped with devices for mixing, aeration, nutrient supply, monitoring and control of process parameters ( pH, temperature, dissolved oxygen tension, back pressure, etc.). The behavior of an expression system in a laboratory method does not allow one to predict the behavior of this system in the complex environment of a bioreactor.

The expression systems of the present invention can advantageously be used for the production of recombinant agents on an industrial scale (both in terms of volume and technical system) in combination with a culture regime that is based on the supply of nutrients, in particular in a fed-batch process or in continuous or semi-continuous process.

In some embodiments, the method of the invention is a fed-batch process.

Whereas the batch process is a culture mode in which all the nutrients required for cell culture are contained in the original culture medium without additional supply of additional nutrients during fermentation, in a fed-batch process, after the batch phase comes the fed-batch phase , in which one or more nutrients are supplied to the crop by feeding. The goal of nutrient feeding is to increase the amount of biomass (called a high-density cell culture or HCDC process) to also increase the amount of recombinant protein. Although the feeding schedule is critical and important in most cultivation processes, the present invention is not limited to a particular feeding schedule.

The supply of nutrients (feeding) can be carried out continuously or periodically in accordance with methods known in the art. The make-up mode can be preset (i.e. make-up is carried out independently of the actual process parameters), for example, linear constant make-up, linear increase in make-up, step increase in make-up, or following a mathematical function during make-up, for example exponential make-up.

In a preferred embodiment, the method of the invention is a fed-batch process in which the feeding regime is preset according to an exponential function. When using the exponential feeding regime, the specific growth rate μ of the cell population can be preset at a constant level and optimized taking into account maximum expression of the recombinant protein. Control of the recharge rate is based on the desired specific growth rate μ. When a specific medium is used, as described below, growth can be accurately predicted and predetermined by calculating the aliquot of biomass that should be produced based on the input unit of substrate.

In another preferred embodiment, the exponential feed mode can be accompanied at the final stages of cultivation by a linear constant feed.

Another embodiment of the fed-batch process uses a linear constant feed. Linear constant feeding is characterized by a feeding rate (volume of nutrient medium per unit time), which is constant (i.e., unchanged) during certain cultivation phases.

In another embodiment of the fed-batch process, a linear increase in feed is applied. A linear increase in feeding is characterized by the rate of supply of the nutrient medium, following a linear function. Replenishment in accordance with a linear increasing function is characterized by a certain increase in the recharge rate over a certain increase in time.

In another embodiment of the fed-batch method of the present invention, a feedback control algorithm (as opposed to a predetermined feed mode) is used to control the feed. In the closed-loop feeding process, the feeding rate depends on the actual level of a certain cultivation parameter. Cultivation parameters suitable for closed-loop feeding are, for example, biomass (and the resulting chemical or physical parameters), dissolved oxygen, respiratory coefficient, pH or temperature. Another example of a closed-loop feeding regime is based on the actual glucose concentration in the bioreactor.

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In another embodiment, bacterial cells harboring a genome-based expression cassette according to the present invention are cultured continuously. The continuous fermentation process is characterized by a specific, constant and continuous rate of supply of fresh culture medium into the bioreactor, resulting in the culture medium being simultaneously removed from the bioreactor at the same specific, constant and continuous removal rate. By maintaining the nutrient medium, feed rate, and removal rate at the same constant level, the parameters and culture conditions in the bioreactor remain constant (the so-called steady state). The specific growth rate μ can be preset and is solely a result of the feed rate and volume of growth medium in the bioreactor. Because cells having one or more genome-based expression cassettes are genetically very stable (in contrast to structurally and segregationally unstable plasmid-based expression systems or expression systems in which the cassette inserted into the genome depends on genomic amplification), the number of cell generations ( cell doublings) according to the invention is theoretically unlimited, and therefore the cultivation time. The advantage of culturing a genetically stable genome-based expression system in a continuous manner is that a higher total amount of recombinant protein can be obtained over a period of time compared to genetically unstable prior art systems. In addition, due to the theoretically unlimited culture time, continuous cell culture of the invention can result in a higher total amount of protein over a period of time, even compared to fed-batch culture processes. The non-limiting worked examples below demonstrate the high stability and productivity of the genome-based expression construct.

Another preferred embodiment relates to semi-continuous cell culture. A semi-continuous cultivation process in the context of the invention is a process that is used in the first step as a fed-batch process (ie a batch fermentation step followed by a fed-batch step). Once a certain volume or biomass has been reached (ie, typically the upper limit of the fermenter volume), a significant portion of the cell medium containing the recombinant protein of interest is removed from the bioreactor. Subsequently, feeding is resumed until the biomass or volume of the culture medium again reaches a certain value. This method (removing the culture medium and refilling with recharge) can be carried out at least once and for a theoretically indefinite time.

There are no restrictions regarding the type of culture medium used in the fermentation process. The cultural environment can be semi-definite, i.e. may contain complex media components (eg, yeast extract, soy peptone, casamino acids, etc.), or it may be chemically defined without any complex compounds.

A specific medium is preferably used. Defined media (also called minimal or synthetic media) consists solely of chemically defined substances, i.e. carbon sources such as glucose or glycerol, salts, vitamins and, taking into account possible auxotrophy by strain, specific amino acids or other substances such as thiamine. Most preferably, glucose is used as the carbon source. Typically, the carbon source in the growth medium is a growth-limiting component that controls the specific growth rate.

Significantly higher yields are obtained in the methods of the invention because bacterial growth and a high but physiologically acceptable rate of recombinant gene expression can be maintained throughout the entire production process.

As described above, in the most preferred embodiment of the invention, the protein of interest is under the control of an inducible or controllable promoter.

There are no restrictions on the manner in which protein expression is induced. For example, the inducer can be added as a single or multiple bolus or through continuous refill, the latter also known as inducer refill (induction). There are no restrictions regarding the point in time at which induction occurs. The inducer can be added at the beginning of cultivation or at the point at which continuous nutrient feeding begins or after feeding begins. Inducer feeding can be achieved either by using an inducer contained in the culture medium or by a separate feeding.

The advantage of feeding with an inductor is that it allows you to control the dosage of the inductor, i.e. allows the dosage of a specific or constant amount of inducer to be maintained for a constant number of genes of interest in the production system. For example, inductor feeding allows the inducer to be dosed in a manner that is proportional to the biomass, resulting in a constant inductor to biomass ratio. Biomass units on which the inducer dose may be based may be, for example, cell dry weight (CDW), cell wet weight (WCW), optical density, total cell number (TCN; cells per volume), or colony forming units (CFU per volume) , or online controlled signals that are proportional to biomass (eg fluorescence, turbidity, dielectric constant, etc.). Essentially the method according to the invention

- 41 044676 allows precise dosing of the inductor according to any parameter or signal that is proportional to the biomass, regardless of whether the signal is measured offline or online. Since the number of genes of interest is determined and is a constant unit for biomass (one or more genes per cell), the consequence of this induction regime is a constant dose of inducer per gene of interest. As an additional advantage, the exact and optimal dosage of the amount of inducer relative to the amount of biomass can be determined and optimized experimentally.

It may not be necessary to determine the actual biomass level by analytical methods. For example, it may be sufficient to add an inducer in an amount that is based on previous cultivations (historical biomass data). In another embodiment, it may be preferable to add the amount of inducer per unit of biomass as theoretically calculated or predicted. For example, for fed-batch cultivation (eg, batch or continuous), it is well known that one unit of a growth-limiting component in the growth medium, typically a carbon source, will result in a certain amount of biomass. For example, 1 g of glucose as a growth-limiting substrate will result in approximately 0.33 g of cell dry mass (also expressed by substrate yield coefficient Yx7 _s = 0.33). Therefore, a certain dosage of inducer per gene of interest can also be achieved by a certain dosage of inducer per unit of growth-limiting component, since a certain unit of growth-limiting component leads to a certain unit of biomass, and a certain unit of biomass contains a certain number of molecules of proteins of interest according to the method of invention.

A significant benefit is that feeding a limited amount of inducer prevents metabolic stress and reduces stress in favor of maximizing protein synthesis capacity.

The ratio of inducer to biomass (or gene or growth-limiting substrate) may not necessarily be constant. It can also be linear increase, linear decrease, increase or decrease according to exponential or other mathematical functions, etc. An essential feature of the invention is that the dose of the inducer per gene of interest is determined.

In some embodiments, the method of the invention is a fed-batch process in which the inducer is present in the loaded medium from the start of the culture.

The mode of induction of expression may also be constitutive, meaning that induction is not triggered by chemical or other stimuli, but is constant from the start of culture. Constitutive induction is the preferred induction mode for continuous culture, but is also useful for fed-batch culture.

Recombinant bacteria and methods for producing HMO are well known (see, for example, Priem B. et al. (2002), Glycobiology, 12(4):235-40; Drouillard S. et al. (2006), Angew. Chem. Int ., ed., 45:1778 -1780; Fierfort N. & Samain E. (2008), J. Biotechnol., 134:261-265; Drouillard S. et al. (2010), Carbohydrate Research, 345, 1394- 1399; Gebus S. et al. (2012), Carbohydrate Research, 363, 83-90).

Following the methods described in the art, and according to the invention, the bacterial host can use the endogenous or exogenous guanosine diphosphate (GDP-fucose) synthesis pathway to produce fucosylated HMOs. The GDP-fucose synthesis pathway refers to the sequence of reactions, usually controlled and catalyzed by enzymes, that leads to the synthesis of GDP-fucose. An approximate route for the synthesis of GDP-fucose in E. coli is outlined below. In this synthesis pathway, enzymes for the synthesis of GDP-fucose include 1) manA=phosphomannose isomerase (PMI); 2) manB=phosphomannomutase (PMM); 3) manC=mannose-1-phosphate-guanylytransferase (GMP); 4) gmd=GDR-mannose-4,6 dehydratase (GMD); 5) fcl=GDP-fucose synthase (GFS); and 6) ΔwcaJ=mutated UDP-glucoselipid carrier transferase.

Glucoseα^Glc-6-P^Pru-6-P^ ^l Mαn-6-P^ ² Mαn-1-P^3GDP-Mαn^4 5GDP-Fuc ⁶ Colanoic acid

The pathway for the synthesis of GDP-fucose from fructose-8-phosphate, a common metabolic intermediate in all organisms, consists of 5 enzymatic steps: 1) PMI (phosphomannose isomerase); 2) PMM (phosphomannomutase); 3) GMP (mannose-1-phosphate-guanylytransferase); 4) GMD (GDP-mannose-4,6-dehydratase); and 5) GFS (GDP-fucose synthase). In the context of the present invention, enzymes of the GDP synthesis pathway, which contribute to increasing the intracellular pool of GDP-fucose, are included in the group of beneficial proteins that are indirectly involved in the synthesis of HMO. Individual bacterial species have different genetic capabilities for the synthesis of GDP-fucose. For example, E.coIi can synthesize enzymes that are capable of performing all five steps, while Bacillus licheniformis lacks enzymes that are capable of performing steps 4 and 5 (i.e., GMD and GFS). Any enzymes in the GDP synthesis pathway that are not naturally present in any particular bacterial species can be introduced into the host by molecular engineering using the recombinant DNA constructs of the invention. Genes encoding the missing enzymes can be presented either in a plasmid expression vector or as exogenous genes integrated into the host chromosome (enzymes of the synthesis pathway

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GDPs in the context of the invention are enzymes that are indirectly involved in the production of HMOs, i.e. are enzymes necessary for the production of HMO).

A bacterium suitable for HMO production, for example E. coli, may contain an endogenous β-galactosidase gene or an exogenous β-galactosidase gene, for example E. coli contains an endogenous lacZ gene (for example, with GenBank accession number V00296 (GI: 41901)). For purposes of the invention, an HMO-producing bacterial cell is genetically modified to either include any β-galactosidase gene or to include an inactivated gene. A gene can be inactivated by completely or partially deleting the corresponding nucleotide sequence from the bacterial genome, or the gene sequence is mutated so that it is not transcribed at all, or is not translated when transcribed, or is translated into a protein (i.e., β-galactosidase), a protein would not have the corresponding enzymatic activity. Thus, the HMO-producing bacterium accumulates an increased intracellular pool of lactose, which is beneficial for HMO production.

A functional lactose permease gene is preferably present in the HMO-producing bacterium of the invention. The lactose permease gene is an endogenous lactose permease gene or an exogenous lactose permease gene. For example, the lactose permease gene contains the lacY gene from E. coli (eg, GenBank accession number V00295 (GI:41897)). Many bacteria have the genetic ability to transport lactose from the culture medium into the cell, using a transport protein that is either a homolog of E. coli lactose permease (for example, found in Bacillus licheniformis) or a transporter that is a member of the common PTS family of sugar transporters (for example, found in in Lactobacillus casei and Lactobacillus rhamnosus). For bacteria that do not have the genetic ability to transport extracellular lactose into the cell cytoplasm, this ability is provided by an exogenous lactose transporter gene (e.g., E. coli lacY) provided on recombinant DNA constructs and delivered either in a plasmid expression vector or as exogenous genes integrated into the host chromosome.

To produce fucosylated oligosaccharide by biosynthesis, the bacterium preferably contains a mutation in the gene for the synthesis of endogenous colanic acid (a fucose-containing exopolysaccharide). By colanic acid synthesis gene is meant a gene involved in the sequence of reactions, typically controlled and catalyzed by enzymes, that lead to the synthesis of colanic acid. Typical colanic acid synthesis genes include the rcsA gene (e.g. GenBank accession no. M58003 (GI:1103316)), the rcsB gene (e.g. GenBank accession no. E04821 (GI:2173017)), the wcaJ gene (e.g. GenBank accession - (amino acid sequence) BAA15900 (GI:1736749), wzxC gene, (e.g. GenBank accession number - (amino acid sequence) BAA15899 (GI:1736748)), wcaD gene, (e.g. GenBank accession number - ( amino acid sequence) BAE76573 (GI:85675202)), wza gene, (e.g. GenBank accession number - (amino acid sequence) BAE76576 (GI:85675205)), wzb gene (e.g. GenBank accession number - (amino acid sequence) BAE76575 ( GI:85675204)), and the wzc gene (eg, GenBank accession number - (amino acid sequence) BAA15913 (GI:1736763)).

This is achieved through a series of genetic modifications of endogenous E. coli genes involved either directly in the biosynthesis of the colanic acid precursor or in the overall control of the synthetic colanic acid regulon. In particular, the ability of the E. coli host strain to synthesize the extracellular capsular polysaccharide, colanic acid, is eliminated by deletion of the wcaJ gene encoding the UDP-glucose transferase to a lipid carrier. In the absence of wcaJ, GDP-fucose accumulates in the cytoplasm of E. coli. Overexpression of the positive regulatory protein, RcsA, in the colanic acid synthesis pathway results in increased intracellular levels of GDP-fucose. Overexpression of an additional positive regulator of colanic acid biosynthesis, namely RcsB, is also used instead of or in addition to overexpression of RcsA to increase intracellular levels of GDP-fucose. Alternatively, colanic acid biosynthesis is increased following the introduction of a null mutation in the lon gene from E. coli (eg, GenBank accession number L20572 (GI:304907), incorporated herein by reference). Lon is an adenosine 5'-triphosphate (ATP)-dependent intracellular protease that is responsible for the degradation of RcsA, which was mentioned above as a positive transcriptional regulator of colanic acid biosynthesis in E. coli. Against the background of zero expression of lon protease, RcsA stabilizes, the level of RcsA increases, the expression of genes responsible for the synthesis of GDP-fucose in E. coli increases (i.e., they are overexpressed), and the intracellular concentration of GDP-fucose increases. The RcsA and RcsB proteins are considered useful for the purposes of the invention, and in some embodiments, their levels in host cells are increased using constructs of the invention wherein the corresponding genes are operably linked to a glp promoter, preferably PglpF.

Preferably, the bacterium producing fucosylated HMG contains an exogenous fucosyltransferase gene. For example, the exogenous fucosyltransferase gene encodes α(1,2)-fucosyltransferase and/or α(1,3)-fucosyltransferase. An example of an a(1,2)-fucosyltransferase gene is the wcfW gene from Bacteroides fragilis NCTC 9343. An example of an a(1,3)-fucosyltransferase gene is the futA gene

- 43 044676 from Helicobacter pylori 26695. One example of the futA gene from Helicobacter pylori is submitted to GenBank under accession number HV532291 (GI:365791177). In the context of the invention, enzymes with fucosyltransferase activity (such as, for example, fucosyltransferases encoded by the above genes) are referred to herein as proteins that are important for the production of fucosylated HMOs and are directly involved in the production of one or more fucosylated HMOs.

The method for producing fucosylated HMOs by biosynthesis in accordance with the invention may include the following steps: providing a bacterium that contains a dysfunctional β-galactosidase gene, an exogenous fucosyltransferase gene, where the exogenous fucosyltransferase gene is part of an expression cassette, where the gene is operably linked to a glp promoter, for example PglpF, a mutation in the colanic acid gene cluster and a functional lactose permease gene; cultivating the bacterium in the presence of a carbon source, such as glycerol, glucose, sucrose, lactose, etc.; and isolating fucosylated HMOs from the bacterium or from a culture supernatant of the bacterium. The HMO-producing bacteria used herein are genetically modified to increase the intracellular pool of lactose (relative to wild type), to increase the level of fucosyltransferase activity and, optionally, to increase the pool of intracellular guanosine diphosphate (GDP)fucose. According to the invention, said bacteria contain at least one nucleotide construct, which contains a nucleotide sequence encoding an enzyme that is directly or indirectly involved in the production of HMO, and a glp promoter according to the invention, preferably PglpF, which is operably linked to this nucleotide sequence. The bacterium may also contain a mutation in a colanic acid (fucose-containing exopolysaccharide) pathway gene, such as the wcaJ gene, resulting in an increased intracellular pool of GDP-fucose. The endogenous E. coli lacZ gene is preferably deleted or functionally inactivated, but in such a way that the expression of the downstream lactose permease gene (lacY) remains unchanged. An organism modified as described above retains the ability to tolerate lactose from the culture medium and creates an intracellular pool of lactose for use as a sink sugar in oligosaccharide synthesis. The bacterium may further contain an exogenous rcsA and/or rcsB gene (for example, in an ectopic nucleotide construct such as a plasmid), and the bacterium optionally further contains a mutation in the 1acA gene

Bacteria having fucosyltransferase activity may include one or both of an exogenous fucosyltransferase gene encoding a(1,2)-fucosyltransferase and an exogenous fucosyltransferase gene encoding a(1,3)-fucosyltransferase. An example of an α(1,2)-fucosyltransferase gene is the wcfW gene from Bacteroides fragilis NCTC 9343. Other α(1,2)-fucosyltransferase genes that use lactose as an acceptor sugar (for example, the futC gene from Helicobacter pylori 26695 or the wbsJ gene from E. coli 0128: B12) can be easily replaced by the wcfW gene from Bacteroides fragilis. One example of the futC gene from Helicobacter pylori is provided under GenBank accession number EF452503 (GI:134142866). An example of an α(1,3)-fucosyltransferase gene is the futA gene from Helicobacter pylori 26695, although other α(1,3)-fucosyltransferase genes known in the art may be substituted (e.g., α(1,3)-fucosyltransferase genes from Helicobacter hepaticus Hh0072, Helicobacter bills, Campylobacter jejuni or from Bacteroides species). Some examples of a(1,3)-fucosyltransferases and other enzymes that are involved in the production of various fucosylated HMOs are shown in Table. 4 below.

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Table 4

Gene Original species Access number Enzyme Example of CME MAMA_R764 Mimivirus Acanthamoeba polyphaga AGC02224.1 α—1,3- fucosyltransferase 2'FL, 3'FL, DFL, LNFP-I, LNFP-III, LNFP-V, LNFP-VI, LNDFH-II, FpLNHI Mg791 Megavirus chiliensis AEQ33441.1 α—1,3- fucosyltransferase 2'FL, 3'FL, DFL, LNFP-I, LNFP-III, LNFP-V, LNFP-VI, LNDFH-II, FpLNHI Moumou_00703 Mimivirus Acanthamoeba polyphaga M10A AGC02224.1 α—1,3- fucosyltransferase 2'FL, 3'FL, DFL, LNFP-I, LNFP-III, LNFP-V, LNFP-VI, LNDFH-II, FpLNHI futA Helicobacter pylori ATCC 26695 NP_207177.1 α—1,3- fucosyltransferase 2'FL, 3'FL, DFL, LNFP-I, LNFP-III, LNFP-V, LNFP-VI, LNDFH-II, FpLNHI fucT Helicobacter pylori NCTC 11639 (short) AAB81031.1 α—1,3- fucosyltransferase 2'FL, 3'FL, DFL, LNFP-I, LNFP-III, LNFP-V, LNFP-VI, LNDFH-II, F- pLNHI ysTSh Helicobacter pylori ATCC 43504 AY450598.1 α—1,4- fucosyltransferase LNDFH-I, LNDFH-II fucTa Helicobacter pylori UA948 AF194963.2 α—1.3/4- fucosyltransferase LNFP-II LNDFH-I LNDFH-II

The bacterium may contain an expression cassette of the present invention providing expression or overexpression of one or more of the above fucosyltransferases and, accordingly, higher production of one or more fucosylated HMGs, for example 2'-FL, 3FL, DFL,

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LNFP-I, -II, -III, -V, VI, LNDFH-I, -II or -Ш.

The invention, in additional embodiments, relates to HMO-producing host cells that contain one or more nucleotide constructs comprising one, two, three, or more of any of the genes described herein (i.e., genes encoding important or beneficial proteins for HMO production), where preferably at least one of the constructs contains a Pglp promoter operably linked to at least one of the genes, where at least one of the constructs is integrated into the genome, where preferably at least one of the constructs is integrated into genome, contains a Pglp promoter operably linked to at least one of the genes encoding a protein that is necessary for the synthesis of HMO, and this construct is present in the genome in low copy number. In some embodiments, the host cells of the present invention may produce fucosylated HMOs, in other embodiments, the cells may produce stalled HMOs, and in other embodiments, the cells may produce non-fucosylated neutral HMOs.

To prepare the sialylated HMOs described herein, general principles and methods previously described in the art can be used (see, for example, Drouillard S. et al. (2010), Carbohydrate Research, 345:1394-1399; or Fierfort N . & Samain E. (2008), J. Biotechnol., 134:261-265).

In general, an engineered bacterial cell that is capable of producing a stylated human milk oligosaccharide, such as 6'-SL (6'-sialyllactose), contains an exogenous sialyltransferase gene encoding a(2,6)-stalyltransferase. The bacterial cell may be E. coli. The exogenous sialyltransferase gene used to produce the 6'-SL can be obtained from any available sources, for example those described for a number of organisms of the genus Photobacterium. Another sialylated HMO, such as 3'-SL (3'-sialyllactose), can be produced by engineered bacteria containing an exogenous nucleic acid molecule encoding an α(2,3)sialyltransferase. The exogenous sialyltransferase gene used to produce the 3'-SL can be obtained from any available source, for example, from Neisseria meningitidis and Neisseria gonorrhoeae. Some examples of suitable sialyltransferases are listed in Table. 5 below.

Table 5

Gene Original species Access number Enzyme Example of CME Pd2.6ST Photobacterium damselae JT0160 BAA25316.1 α-2,6 sialyltransferase 6'SL PspST6 Photobacterium sp. JT-ISH-224 BAF92026.1 α—2,6- sialyltransferase 6'SL PiST6_145 Photobacterium leiognathi JT-SHIZ145 BAF91416.1 α—2,6- sialyltransferase 6'SL PiST6_119 Photobacterium leiognathi JT-SHIZ119 BAI49484.1 α—2,6- sialyltransferase 6'SL NST Neisseria meningitidis MC58 Neisseria AAC44541.1 α—2,3- sialyltransferase 3'SL NGO_1081 gonorrhoeae (strain ATCC 700825/FA1090) YP_208160.1 α—2,3- sialyltransferase 3'SL

Preferably, the engineered bacterium contains a deficient (impaired) sialic acid catabolic pathway. The sialic acid catabolic pathway refers to the sequence of reactions, usually controlled and catalyzed by enzymes, that lead to the degradation of sialic acid. A typical sialic acid catabolic pathway described herein is the E. coli pathway. In this pathway, sialic acid (Neu5Ac; N-acetylneuraminic acid) is degraded by the enzymes NanA (N-acetylneuraminic acid lyase) and NanK (N-acetylmannosamine kinase) and NanE (N-acetylmannosamine 6-phosphate epimerase), all encoded by the nanATEK- operon. yhcH and are suppressed by NanR (http://ecocyc.org/ECOLI). Sialic acid catabolic pathway deficiency is achieved in the E. coli host by introducing mutations in endogenous nanA (N-acetylneuraminate lyase) (e.g., GenBank accession number D00067.1(GL216588)) and/or nanK (N-acetylmannosamine kinase) (e.g. GenBank accession number (amino acid sequence) BAE77265.1 (GL85676015)) and/or paPE

- 46 044676 (N-acetylmannosamine b-phosphate epimerase, GI:947745, incorporated herein by reference). It is not necessary that the nanT (N-acetylneuraminate transporter) gene is also inactivated or mutated. Other intermediates of sialic acid metabolism include: (ManNAc-6-P) N-acetylmannosamine-6-phosphate; (GIcNAc-6-P) N-acetylglucosamine-6-phosphate; (GIcN-6-P) Hycosamine 6-phosphate and (Fruc-6-P) fructose 6-phosphate. In some preferred embodiments, nanA is mutated. In other preferred embodiments, nanA and nanK are mutated while nanE remains functional. In another preferred embodiment, nanA and nanE are mutated, while nanK has not been mutated, inactivated or deleted. A mutation is one or more changes in the nucleic acid sequence encoding the gene product nanA, nanK, nanE and/or nanT. For example, a mutation may represent 1, 2, up to 5, up to 10, up to 25, up to 50, or up to 100 changes in the nucleic acid sequence. For example, the nanA, nanK, nanE and/or nanT genes are mutated by a null mutation. Null mutations, as described herein, cover amino acid substitutions, additions, deletions or insertions that either cause loss of enzyme function (ie, reduced or absent activity) or loss of enzyme (ie, absence of gene product). By deletion it is meant that the coding region is removed in whole or in part so that no (functional) gene product is formed. By inactivation it is meant that the coding sequence has been altered such that the resulting gene product is functionally inactive, or the encoded gene product has less than 100%, for example 90, 80, 70, 60, 50, 40, 30 or 20%, of the activity of the native endogenous gene product of natural origin. An unmutated gene or protein does not differ from the native, naturally occurring, or endogenous coding sequence by 1, 2, up to 5, up to 10, up to 20, up to 50, up to 100, up to 200, or up to 500 or more codons or up to the length of the corresponding encoded amino acid sequences.

In addition, the bacterium (eg E. coli) also has the ability to synthesize sialic acid. For example, a bacterium has the ability to synthesize sialic acid due to the presence of exogenous UDP-GlcNAc 2-epimerase (for example, neuC from Campylobacter jejuni (GenBank AAK91727.1; GL15193223) or equivalent (for example, neuC from E. coli S88 (GenBank YP_002392936.1 ; GI: 218560023), Neu5Asynthase (e.g., neuB from C. jejuni (GenBank AAK91726.1; GI: 15193222) or equivalent (e.g., sialic acid synthase from Flavobacterium limnosediminis, GenBank GL559220424) and/or CMP-Neu5Asynthetase (e.g. neuA from C. jejuni (GenBank AAK91728.1; GI: 15193224) or equivalent (e.g., CMP-sialic acid synthase from Vibrio brasiliensis, GenBank GI: 493937153).

Bacteria that produce sialylated HMOs contain one or more exogenous sialyltransferases that are encoded by the DNA-encoding expression cassette of the invention, which is present in host cells either as a plasmid or integrated into the genome. Preferably, at least one of the one or more DNA sequences encoding the sialyltransferase is operably linked to the glp promoter described herein, preferably PglpF. Non-limiting examples of suitable sialyltransferases are given in table. 5.

A bacterium capable of synthesizing sialic acid may preferably be engineered to have increased production of UDP-GlcNAc. A typical way to achieve this is to overexpress the positive endogenous regulator of UDPGlcNAc synthesis, such as simultaneous overexpression of the E. coli nagC and glmS genes. This overexpression of nagC and glmS is preferably achieved by operably linking the genes to the glp promoter of the invention and expressing the cassette as integrated into the genome, or alternatively it can be achieved by providing additional copies of the nagC and glmS genes linked to glp or another promoter in a plasmid vector.

The production of neutral N-acetylglucosamine-containing HMOs in engineered bacteria is also known in the art (see, for example, Gebus C. et al. (2012), Carbohydrate Research, 363, 83-90).

For the production of N-acetylglucosamine-containing HMOs, such as lacto-N-triose 2 (LNT2), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentase I (LNFP-)I), lacto- N-fucopentase II (LNFP-II), lacto-M-fucopentase III (LNFP-III), lacto-M-fucopentase V (LNFP-V), lacto-M-difucohexase I (LDFH-I), lacto-N- difucohexaose II (LDFH-II) and lacto-N-neodifucohexaose II (LNDFH-111), the bacterium contains a functional lacY and a dysfunctional lacZ gene as described above, and it is engineered to contain an exogenous UDP-GlcNAc:Galα/β gene -Rβ3-N-acetylglucosaminyltransferase or a functional variant or fragment thereof. This exogenous UDP-GlcNAc:Gala/e-Re3N-acetylglucosaminyltransferase gene can be obtained from any of a number of sources, for example from the lgtA genes described for N. meningitides (Genbank, protein accession number AAF42258.1) or N. gonorrhoeae (Genbank , protein accession number ACF31229.1). Optionally, an additional exogenous glycosyltransferase gene can be coexpressed in a bacterium containing an exogenous UDP-GlcNAc:Gala/βRβ3-N-acetylglucosaminyltransferase. For example, the β-1,4-galactosyltransferase gene is coexpressed with the UDP-GlcNAc:Galα/β-Rp3-N-acetylglucosaminyltransferase gene. This exogenous β-1,4 galactosyltransferase gene can be obtained from any of a number of sources, such as those described for

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N. meningitidis gene IgtB (Genbank, protein accession number AAF42257.1) or the gene described for H. pylori HP0826/galT (Genbank, protein accession number NP 207619.1). Optionally, the additional exogenous glycosyltransferase gene coexpressed in bacteria containing the exogenous UDP-GlcNAc:Gala/eRp3-N-acetylglucosaminyltransferase gene is a β-1,3-galactosyltransferase gene, such as that described for E. coli 055:H7, the wbgO gene ( Genbank, protein accession number YP003500090.1), or for H. pylori gene jhp0563 (Genbank, protein accession number AEZ55696.1), or for Streptococcus agalactiae type Ib OI2 gene cpslBJ (Genbank, protein accession number AB050723). Functional variants and fragments of any of the enzymes described above are also covered by the present invention.

Preferably, at least one gene encoding an enzyme as any of the above, i.e. both/any N-acetylglucosaminyltransferase gene and/or galactosyltransferase gene were operably linked to the Pglp of the invention and expressed from the corresponding genomically integrated cassette. In one embodiment, the gene that is integrated into the genome is a gene encoding a galactosyltransferase, for example the HP0826 gene encoding the GalT enzyme from H. pylori (Genbank, protein accession number NP 207619.1); in another embodiment, the gene that is integrated into the genome is a gene encoding a β-1,3-N-acetylglucosaminyltransferase, for example the lgtA gene from N. meningitides (Genbank, protein accession number AAF42258.1). In these embodiments, the second gene, i.e. the β-1,3-N-acetylglucosaminyltransferase or galactosyltransferase gene, respectively, can be expressed from either a genome-integrated or plasmid cassette. The second gene may optionally be expressed either under the control of a glp promoter or under the control of any other promoter suitable for the expression system, for example Plac.

Advantageously, bacteria producing N-acetylglucosamine-containing HMOs can be engineered to have an increased intracellular pool of UDP-GlcNAc. A typical means of achieving this trait is overexpression of the positive endogenous regulator of UDP-GlcNAc synthesis, such as simultaneous overexpression of the E. coli nagC and glmS genes. This overexpression of nagC and glmS is preferably achieved by operatively linking the genes to the glp promoter of the invention and integrating the cassette into the host genome, or alternatively this can be achieved by providing additional copies of the nagC and glmS genes linked to glp or another promoter in the plasmid vector.

For HMO production, HMO-producing bacteria described herein are cultured according to procedures known in the art in the presence of a suitable carbon source, such as glucose, glycerol, lactose, etc., and the resulting HMO is collected from the culture medium. and microbial biomass formed during the cultivation process. The HMOs are then purified according to procedures known in the art, such as those described in WO 2015188834, WO 2017182965 or WO 2017152918, and the purified HMOs are used as nutraceuticals, pharmaceuticals or for any other purpose, such as research .

Other features and advantages of the invention will be apparent from the following description of working examples and from the claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention relates. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods and examples are illustrative only and therefore do not limit the scope of the invention.

Examples

Materials and methods.

Unless otherwise noted, standard techniques, vectors, control sequence elements, and other expression system elements known in the art of molecular biology are used to manipulate, transform, and express nucleic acids. Such standard methods, vectors and elements can be found, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology (1995) (John Wiley &Sons); Sambrook, Fritsch & Maniatis (eds.), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology, 152: Guide to Molecular Cloning Techniques (1987) (Academic Press); Bukhari et al. (eds.), DNA Insertion Elements, Plasmids and Episomes (1977) (Cold Spring Harbor Laboratory Press, NY); Miller, J.H., Experiments in molecular genetics (1972) (Cold spring Harbor Laboratory Press, NY).

Strains and plasmids.

The bacterial strain used, MDO, was constructed from Escherichia coli K12 DHL E. coli K12 DH1 genotype: F ^- , λ ^- , gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. In addition to E. coli K12 DH1, the MDO genotype has the following modifications: lacZ: 1.5 kb deletion, 1acA 0.5 kb deletion, nanKETA 3.3 k deletion mdoH mc/oH: deletion of 0.5 kb, and insertion of the Plac promoter upstream of the gmd gene.

The strains used in the present examples are described in table. 6. Donor and auxiliary

- 48 044676 plasmids used to construct these strains are included in table. 7 along with high copy number plasmids introduced into some of the constructed strains.

Table 6

Source strains Strain identifier, ID Genome description Description of the plasmid DH1 F“ 2' endAl recAl relAl gyrA96 thi1 glnV44 hsdR17(r _K Шк) MDO E coli DH1 JlacZ, JlacA JnanKETA zimelA dwcaJ, JmdoH Strains expressing reporter genes Strain identifier, ID Genome Description Description of the plasmid MAR8O8 MDO galK::PglpF-lacZ-Tl - MAR1010-9 MDO galK::PglpF400-9-lacZ-Tl - MAR1010-11 MDO galK::PglpF400-ll-lacZ-Tl - MAR1010-13 MDO galK::PglpF400-13-lacZ-Tl - MAR1010-17 MDO galK::PglpF400-17-lacZ-Tl - MAR1010-19 MDO galK::PglpF400-19-lacZ-Tl - MAR1010-20 MDO galK::PglpF400-20-lacZ-Tl - MAR1025 MDO galK::PglpA-lacZ-Tl - MAR1026 MDO galK::PglpD4acZ-Tl - MAR1027 MDO galK::PglpT-lacZ-Tl - MAP1086 MDO galK::Δ 175PglpF-lacZ-Tl - MAPI 176 MDO galK::PglpF_SDl-lacZ-Tl - MAPI 178 MDO galK::PglpF_SD3-lacZ-Tl - MAPI 179 MDO galK::PglpF_SD4-lacZ-Tl - MAP 1180 MDO galK::PglpF_SD5-lacZ-Tl - MAP1181 MDO galK::PglpF_SD6-lacZ-Tl - MAP 1182 MDO galK::PglpF_SD7-lacZ-Tl - MAPI 183 MDO galK::PglpF_SD8-lacZ-Tl - MAP 1184 MDO galK::PglpF_SD9-lacZ-Tl - MAPI 185 MDO galK::PglpF_SD10-lacZ-Tl - MAP1206 MDO galK::PglpF_SD2-lacZ-Tl - MAP1209 MDO galKMOPglpFAacZ-Tt - MAP1210 MDO galK:A25PglpF-lacZ-Tl - MAP1211 MDO galK:A150PglpF-\acZD/\ - MAP1356 MDO galK::PglpF-lacZ-Tl-galK glpR::kanR — MAP 13 65 MDO galK::PglpA_org-lacZ-TlgalK — MAP 13 66 MDO galK::PglpD_org-lacZ-TlgalK — MAP 13 67 MDO galK::PglpT_org-lacZ-TlgalK —

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MAP 13 68 MDO galK::PglpF_org-lacZ-TlgalK — MAP 1370 MDO galK::Plac_org-lacZ-Tl-galK — Strains expressing reporter genes Strain identifier, ID Genome description Description plasmids MAP219 MDO Plac-Pd2 - MAP700 MDO Plac-nst - MAP710 MDO PglpF-nst - MAR986 MDO PglpF-Pd2 - MDO1 MDO pBBR3-Plac-lgtA-tet, pBS-Plac-galT-amp MDO15 MDO pBBR3-Plac-lgtA-tet, pBS-Plac-galTK-amp MP166 MDO 3xPlac-lgtA3xPlac-galT lad::CP6-galK - MP245 MDO 3xPlac-lgtA2xPlac-gaZ7X Alaci - MP1497 MDO PglpF-lgtA pBS-Plac-galT-amp MP1498 MDO PglpF-lgtA pBS-Plac-galTK-amp MP1499 MDO PglpF-galT pBBR3-Plac-lgtA-tet MP1655 MDO 2xPglpF-galTK pBBR3-Plac-lgtA-tet MP1825 MDO PglpF-galT PglpF-lgtA lad: :CP6-galK - MP1920 MDO PglpF-galTK PglpF-IgtA lad::CP6-galK - MP2239 MDO PglpF-galTK PglpF-lgtA PglpF-futC Alaci - MP2374 MDO PglpF-galTK PglpF-lgtA PglpF-futC PglpF-SL Alaci - MP2622 MDO Plac-lgtAPlac-galT - MAP265 MDO Plac-Pd2 nadC: :galK pBS-Plac-neuBCAnadC MAP425 MDO 2xPlac-nst Plac-neuBCA A50lacI pBS-Plac-neuBCAnadC MAR1200 MDO PglpF-neuA PglpF-neuB PglpF-neuC PglpF-Pd2 - MAR1214 MDO PglpF-neuA PglpF-neuB PglpF-neuC - PglpF-nst FT18 MDO pBP-Plac-futC-kan pBBR3-Plac-gmd-fdcpsB-cpsG MAR965 MDO PglpF-gmd-fd-grnm-wcal-cpsB-cpsG PglpF-futC_op -

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Table 7

Plasmid identifier, ID Description pACBSR Para-LSceI-2 Red, pl5A ori, cam* pUC57 pMBl, bla pUC57::gal pUC57::galTK' / Tl-galKM' pMAP99 pVC57-galTK '-Plac-Pd2_op-Tl -galKM' pMAP205 p UC5 7: : galTK ’-PglpF -lacZ-Tl -galKM’ pMAP216 pUC57-galTK '-P1 ac-J29nst_op-T 1 -galKM' pMAP228 pUC57-ga/TK '-PglpF-J29nst_op-Tl -galKM' pMAP391 pUC57-ga/TX’-PglpF-Pd2-Tl-ga/XM’ pMAP431 pVC57-galTK '-PglpA-lacZ-Tl -galKM' pMAP432 pVC57-galTK '-PglpD-lacZ-Tl -galKM' pMAP433 pUC57 -galTK '-PglpT-lacZ-Tl -galKM' pMAP457 pUC57-ga/TX’-D25PglpF-lacZ-Tl-ga/^ pMAP462 pVC57-galTK '-D150PglpF-lacZ-Tl -galKM' pMAP463 pUC57 -galTK '-D 175PglpF-lacZ-Tl-ga/XM' pMAP486 pUC57-ga/7X '-PglpF_SD 1 -lacZ-Tl -galKM' pMAP487 pUC57-galTK '-PglpF_SD2-lacZ-Tl -galKM' pMAP488 pUC57-ga/TX’-PglpF_SD3-lacZ-Tl-ga/XM’ pMAP489 pVCM-galTK '-PglpF_SD4-lacZ-Tl -galKM' pMAP490 pVC57-galTK '-PglpF_SD5-lacZ-Tl -galKM' pMAP491 pUC57-ga/TK '-PglpF_SD6-lacZ-Tl -galKM' pMAP492 pVC57-galTK '-PglpF_SD7-lacZ-Tl -galKM' pMAP493 pUC57-ga/TK '-PglpF_SD8-lacZ-Tl -galKM' pMAP494 pUC57-ga/7X’-PglpF_SD9-lacZ-Tl -galKM’ pMAP495 pUC57 -galTK '-PglpF_SD 10-lacZ-Tl-ga/XM' pMAP537 p\JC57-galTK '-D190PglpF-lacZ-Tl -galKM' pMAP689 pVC57-galTK '-PglpA_org-lacZ-Tl -galKM' pMAP690 pVC57-galTK '-PglpD_org-lacZ-Tl -galKM' pMAP691 pUC57-galTK '-PglpT_org-lacZ-Tl -galKM' pMAP693 pVC57-galTK '-PglpF_org-lacZ-Tl -galKM' pMAP695 pVC57-galTK '-Plac_org-lacZ-Tl -galKM' MP55 pBBR3-Plac-lgtA-tet MP46 pBS-Plac-galT-amp MP139 pBS-Plac-galTK-amp pMAPlOl pBS-Plac-neuBCA-nadC MP415 pBP-Plac-futC-kan MP416 pBBR3-Plac-gmd-fd-cpsB-cpsG

Wednesdays.

Luria Broth (LB) medium was prepared using LB Broth Powder, Millers (Fisher Scientific), and LB agar plates were prepared using LB Agar Powder, Millers (Fisher Scientific). Screening of strains on LB plates containing 5-bromo-4-chloro-3-indolyl-pD-galactopyranoside (X-gal) was performed using an X-gal concentration of 40 μg/ml. With the addition of a suitable antibiotic, ampicillin (100 µg/ml) and/or chloramphenicol (20 µg/ml).

The basal minimal medium had the following composition: NaOH (1 g/l), KOH (2.5 g/l), KH2PO4 (7 g/l), NH4H2PO4 (7 g/l), citric acid (0.5 g/l ), microelement solution (5 ml/l). The concentrated solution of trace elements contained: ZnSO ₄ -7H ₂ O 0.82 g/l, citric acid 20 g/l, MnSO ₄ -H ₂ O 0.98 g/l, FeSO ₄ -7H ₂ O 3.925 g/l, CuSO ₄ -5H ₂ O 0.2 g/l. The pH of the basal minimal medium was adjusted to 7.0 using

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N NaOH and autoclaved. Before inoculation, the basic minimal medium was supplemented with 1 mM MgSO ₄ , 4 μg/ml thiamine, 0.5% of a given carbon source (glucose, glycerol, sorbitol, xylose, lactose, maltose (MgSO ₄ )) and, if necessary, added isopropyl-β- D-thiogalactoside (IPTG) (0.2 mM), ampicillin (100 μg/ml) and/or chloramphenicol (20 μg/ml). Thiamine, antibiotics and IPTG were sterilized by filtration. All percentage concentrations for glycerol are presented as v/v and for glucose, sorbitol, xylose, lactose and maltose as w/v.

M9 plates containing 2-deoxygalactose had the following composition: 15 g/L agar (Fisher Scientific), 2.26 g/L 5x M9 minimal salt (Sigma-Aldrich), 2 mM MgSO ₄ , 4 μg/mL thiamine, 0 .2% glycerol and 0.2% 2-deoxy-D-galαctose (Carbosynth).

MacConkey indicator plates containing galactose had the following composition: 40 g/L MacConkey agar base (BD Difco™). After autoclaving and cooling to 50°C, D-galactose (Carbosynth) was added to a final concentration of 1%.

Cultivation.

Unless otherwise stated, E. coli strains were cultured in Luria-Bertani (LB) medium containing 0.2% glucose at 37°C with agitation.

Cultures collected for β-galactosidase assays were prepared as follows: one colony per LB plate was precultured in 1 ml of minimal basal medium containing glucose (0.5%) in 10 ml of a 24-well deep-well plate ( Deep well plate, Axygen). The plate was sealed before culture with Hydrophobic Gas Permeable Adhesive Seal (Axygen) and incubated for 24 h at 34°C with shaking at 700 rpm in an orbital shaker (Edmund Biihler GmbH). The cell density of the culture was monitored at 600 nm using an S-20 spectrophotometer (Boeso, Germany). 20 μl of the overnight culture was used to inoculate 2 ml of LB medium or basal minimal medium containing glucose or other carbon source (0.5%) in a 24-well deep well plate. Antibiotics and/or IPTG were added as needed. Deep well plates were covered with sealing foil and incubated for 24 hours at 28°C on an orbital shaker at 700 rpm. After incubation, OD ₆₀₀ was measured and 0.5 ml of cell culture was collected by centrifugation for preliminary β-galactosidase assay.

Chemical competent cells and transformations.

E. coli was inoculated from LB plates into 5 ml LB containing 0.2% glucose and cultured at 37°C with shaking to an OD ₆₀₀ of approximately 0.4. 2 ml of culture were collected by centrifugation for 25 s at 13000 g. The supernatant was removed and the cell pellet was resuspended in 600 μl of cold TB solution (10 mM PIPES, 15 mM CaCl ₂ , 250 mM KCl). Cells were incubated on ice for 20 min followed by sedimentation for 15 s at 13,000 g. The supernatant was removed, and the cell pellet was resuspended in 100 μl of cold TB solution. Plasmid transformation was carried out using 100 μl of competent cells and 1-10 ng of plasmid DNA. Cells and DNA were incubated on ice for 20 min and then heat shocked at 42°C for 45 s. After a 2-minute incubation on ice, 400 ml of SOC medium (20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl, 0.186 g/L KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ and 20 mM glucose), and the cell culture was incubated at 37°C with shaking for 1 hour before plating on selective plates.

Plasmid ligations were transformed into chemically competent TOP10 cells under conditions recommended by the supplier (ThermoFisher Scientific).

Methods of working with DNA.

Plasmid DNA from E. coli was isolated using the QIAprep Spin Miniprep kit (Qiagen). Chromosomal DNA from E. coli was isolated using the QIAmp DNA Mini Kit (Qiagen). PNR products were purified using the QIAquick PNR purification kit (Qiagen). DreamTaq PCR Master Mix (Thermofisher), Phusion U hot start PCR master mix (Thermofisher), USER Enzym (New England Biolab) were used according to the supplier's recommendations. Primers were provided by Eurofins Genomics, Germany. PCR fragments and plasmids were sequenced at Eurofins Genomics.

Colony PCR was performed using DreamTaq PCR Master Mix under conditions recommended by the supplier (Thermofisher) in a T100™ Thermal Cycler (Bio-Rad). For example, during the construction of strains expressing a reporter or recombinant gene from the galK locus, primers 048 (5'-CCCAGCGAGACCTGACCGCAGAAC-3') (SEQ ID NO: 58) and 049 β-CCCCAGTCCATCAGCGTGACTACC-3') were used in the colony PCR reaction. (SEQ ID NO: 59) to confirm the correctness of the proposed modification.

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Table 8

Primers used to construct the scaffold used to generate donor plasmids

Name Oligonucleotide sequence, 5'-3' Description S.E. QID NO 040 ATTAACCCUCCAGGCATCAAATAAAACGAAAGGC At ackbone.for 100 079 ATTTGCGCAUCACCAATCAAATTCACCGGGCC Backbone.re ν 101 0261 ATGCGCAAAUGCGGCACGCCTTGCAGATTACG PglpF.for 102 0262 AGCTGTTUCCTCCTTGGTTAATGTTTGTTGTATGC G PglpF.rev 103 068 ATGCGCAAAUTGTGAGTTAGCTCACTCATTAG Plac.for 84 0113 AGCTGTTUCCTCCTTAGGTACCCAGCTTTTGTTCC C Plac.rev 117

Table 9

Heterologous genes included in expression cassettes containing a promoter sequence and an artificial DNA sequence (i) (SEQ ID NO: 70) to enable microbial HMO production

Gene Source of genes Access number Function futC Helicobacter pylori 26695 EF452503 α-1,2-fucosyltransferase IgtA Neisseria meningitidis 053442 CP000381 ΑΕ3-Ν- acetylglucosaminyltransferase galT Helicobacter pylori 26695 AE000511 β-1 D-galactosyltransferase galT Helicobacter pylori Homologous β-1,3-galactosyltransferase TO 43504 BD182026 neuA Campylobacter jejuni ATCC43438 AF400048 CMP Synthetase neuB Campylobacter jejuni ATCC43438 AF400048 Sialic acid synthetase neuC Campylobacter jejuni ATCC43438 AF400048 C\cNx-6-phosphate-2-epimerase nst Neisseria meningitides L3 MC58 U60660 a-2,3-sialyltransferase Pd2 Photobacterim damsela JT0160 VAA25316.1 a-2,6-sialyltransferase

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Table 10

Primers that were used to amplify heterologous genes of interest and ensure compatibility of the resulting PCR products with plasmid scaffolds to enable donor construction

Olignucleotide identifierID Oligonucleotide sequence Description SEQ ID NO MP452 AAACAGCUATGATCTCTGTCTACATCATC AGTCTG galTK_opt for 105 MP453 AGGGTTAAUTGCGCGTTAGACTTCTTTCG GGGTTTTCA galTK_opt rev 106 0123 AAACAGCUATGGCGTTCAAAGTGGTCCAA ATS futC_opt for 107 0124 AGGGTTAAUTGCGCGTTAGCCCAGCGCGT TATATTTCTG futC_opt rev 108 0142 AAACAGCUATGCAACCGCTGGTCTCCGTG C lgtA_opt for 109 0143 AGGGTTAAUTGCGCGTTAACGGTTTTTCA GCAGGCGG lgtA_opt rev 110 0342 AAACAGCUATGTCAAAAGTCGCTCTCATC ACCGG CA for 111 0126 AGGGTTAAUTGCGCGTTACTCGTTCAGCA CA rev 112 ACGTCAGC 0144 AAACAGCUATGCGTGTCTTCGCCATTTCT WITH galT_opt for 113 0145 AGGGTTAAUTGCGCGTTAGACGAATTGCC AGTATTTCAGG galT_opt rev 114 095 AAACAGCUATGGAACGTAACGCCGTGAG CCTGC nst_opt for 115 093 AGGGTTAAUTGCGGCTTAGTTTTTATCGTC AAAGGTCAG nst_opt rev 116 026 AAACAGCUATGTGCAATAGCGATAACACC Pd2_opt for 98 027 AGGGTTAAUTGCGCGTTAGGCCCAGAACA GAACATC Pd2_opt rev 99

Construction of plasmids.

A plasmid was constructed containing two I-Scel endonuclease sites separated by two DNA fragments of the gal operon (required for homologous recombination in galK) and a T1 transcription terminator sequence (pUC57::gal) (GeneScript). The DNA sequences used for homologous recombination in the gal operon covered base pairs 3,628,621-3,628,720 and 3,627,572-3,627,671 in the Escherichia coli K12 MG155 sequence of the complete GenBank genome: ID: CP014225.1. Insertion by homologous recombination will remove 949 bp of galK and the galK phenotype.

Standard techniques well known in the field of molecular biology were used to design primers and amplify specific DNA sequences of Escherichia coli K-12 DHL chromosomal DNA. Such standard techniques, vectors and elements can be found, for example, in: Ausubel et al. (eds.), Current Protocols in Molecular Biology (1995) (John Wiley &Sons); Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) (Academic Press); Bukhari et al. (eds.)

A 3.5 kb plasmid backbone containing pUC57-sceI-galTK-T1-galKM-sceI was amplified using primers 040 (SEQ ID: 7) and O79 (SEQ ID: 8) (Table 8) and DNA fragment size

- 54 044676

3.3 kb containing lacZ was amplified from chromosomal DNA isolated from E. coli K-12 DHL

Chromosomal DNA obtained from E. coli DH1 was used to amplify a 300 bp DNA fragment containing the PglpF promoter (SEQ ID NO: 57) using oligonucleotides 0261 (SEQ ID NO: 102) and 0262 (SEQ ID NO : 103), or the Plac promoter (SEQ ID NO: SEQ ID NO: 11) - using oligonucleotides 068 and 0113 (SEQ ID NO: 84 and 117) (Table 8). Similar to a 107 bp DNA fragment containing the Plac promoter, Plac_org, a 182 bp DNA fragment containing the PglpA promoter, PglpA_org, a 190 bp DNA fragment containing the PglpD promoter, PglpD_org, a 245 DNA fragment bp containing the PglpT promoter, PglpT_org, a 300 bp DNA fragment containing the PglpF promoter, PglpF_org, was amplified from the E. coli DH1 genome.

The sixteen nucleotide sequence located upstream of the translation start site of the glp promoters (SEQ ID NO: 5-8 - glpF, A, D and T, respectively) (including the ribosome binding site) was modified by PNR in the original (org) promoter fragments. Similarly, the Shine-Dalgarno sequence in the PglpF expression element was modified using primers by introducing specific modifications into the oligonucleotides used to amplify DNA fragments, resulting in the promoter expression elements PglpF_SDl, PglpF_SD2, PglpF_SD3, PglpF_SD4, PglpF_SD5, PglpF_SD6, P glpF_SD7, PglpF_SD8 , PglpF_SD9 and PglpF_SD10 (SEQ ID NO: 13-22, respectively).

Shortening of the 5' end of the PglpF expression element was performed using specific primers yielding the promoter expression elements A15PglpF, A140PglpF, A165PglpF and A198PglpF (SEQ ID NOs: 29-32).

All PCR fragments were purified, and the plasmid framework, promoter element (Plac_org, Plac, PglpF_org, PglpF, PglpT_org, PglpT, PglpA_org, PglpA, PglpD_org, PglpD, PglpF_SDl, PglpF_SD2, PglpF_SD3, PglpF_SD 4, PglpF_SD5, PglpF_SD6, PglpF_SD7, PglpF_SD8, PglpF_SD9, PglpF_SD10, A15PglpF, A140PglpF, A165PglpF or A180PglpF) and lacZ were cloned, transformed into TOP10 cells and selected on LB plates containing 100 mg/ml ampicillin and 0.2% glucose. The constructed plasmids (see Table 10) were purified. The promoter sequence and 5′ end of the lacZ gene were confirmed by DNA sequencing (MWG Eurofins Genomics).

Plasmid scaffolds based on pTOPO (ThermoFisher Scientific) or any other plasmid can be prepared in the same manner as described above. All constructed plasmid scaffolds contained two specific DNA fragments homologous to Escherichia coli K-12 DH1, used for homologous recombination. Thus, a genetic cassette containing the PglpF or Plac promoter, any gene of interest, and the T1 transcription terminator was inserted specifically into the Escherichia coli genome. Construction of the plasmids used for recombination was performed using standard cloning methods.

DNA sequences of heterologous genes encoding glycosyltransferases or other enzymes of interest were codon optimized and synthesized using Genescript. Any gene of interest, such as host or heterologous genes, lgtA, galT, galTK, futC, neuA, neuB, neuB, nst or Pd2 (Table 9), can be amplified by PCR using appropriate primers spanning the start codon , ATG, and stop codon, TAA, of the gene (Table 10). For example, the Pd2 gene was amplified using primers 026 (SEQ ID NO: 98) and 027 (SEQ ID NO: 99), while the nst gene was amplified using primers 095 and 093 (SEQ ID NO: 115 and 116) (Table . 9). To construct donor plasmids with these genes, the following procedure was used: a 3.5 kb plasmid backbone containing PglpF (SEQ ID NO: 12) was amplified using pMAP205 as a template. Coding sequences of the Pd2 gene from Photobacterium damselae (JT0160) (see Drouillart et al., 2010, Carbohydrate Research., 345:1394-1399. Efficient synthesis of 6'-sialyllactose, 6,6'-disiallyllactose, and 6'-KDO -lactose by metabolically engeineered E. coli expressing a multifunctional sialyltransferase from the Photobacterium sp. JT-ISH-224) and the nst gene from Neisseria meningitides (MC58) (see Fierfort and Samian, 2008, J. Biotech., 134:261- 265, Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides) were codon optimized for expression in E. coli and synthesized using Genescript. The Pd2 and nst genes were cloned into plasmid scaffolds as described above, which made it possible to obtain plasmids pMAP216, pMAP228, pMAP99, pMAP391 (Table 3). Plasmids were purified, transformed into TOP10 cells, and selected on LB plates containing 100 μg/ml ampicillin and 0.2% glucose. The promoter sequence and 5′ end of the Pd2 nst genes were confirmed by DNA sequencing (MWG Eurofins Genomics).

In general, and for any heterologous gene of interest, all PNR fragments were purified, and the plasmid backbone, glpF promoter element (SEQ ID NO: 54), synthetic DNA sequence (i) (70UTR/SEQ ID NO: 37/synDNA (i); see Table 1) and the gene of interest was cloned using standard USER cloning. Cloning into the appropriate plasmid can be accomplished using any standard DNA cloning technique. After cloning, DNA was transformed into TOP10 cells and selected on LB plates containing 100 μg/ml ampicillin (or 50 mg/ml kanamycin in the case of pTOPO-based constructs) and 0.2% glucose. Constructed plazas

- 55 044676 mids were purified and the promoter sequence and 5' end of the gene of interest were verified by DNA sequencing (MWG Eurofins Genomics).

Strain construction.

Insertion of promoter expression elements fused to a reporter gene or a recombinant gene was performed by Gene Gorging essentially as described in Herring et al. (Herring, C. D., Glasner, J. D., Blattner, F. R. (2003), Gene (311), 153-163). Briefly, donor plasmid and helper plasmid were co-transformed into MD0 and selected on LB plates containing 0.2% glucose, ampicillin (100 μg/ml) or kanamycin (50 mg/ml) and chloramphenicol (20 μg/ml). A single colony was inoculated in 1 ml of LB containing chloramphenicol (20 μg/ml) and 10 μl of 20% L-arabinose and incubated at 37°C with shaking for 7-8 hours. Cells were then plated on plates containing M9-DOG and incubated at 37°C for 48 hours. Single colonies formed on MM-DOG were streaked on LB plates containing 0.2% glucose and incubated for 24 hours at 37°C.

For insertions at the galK locus, colonies that appeared white on MacConkey-galactose agar plates and were sensitive to ampicillin and chloramphenicol were expected to lose the donor and helper plasmid and contain the insertion at the galK loci. Insertions at galK sites were identified by colony PCR using primers 048 and 049 located outside the galK loci. Chromosomal DNA was purified, the galK locus was amplified using primers 048 and 049, and the inserted DNA was verified by sequencing (Eurofins Genomics, Germany). Strains MAP1365, MAP1366, MAP1367, MAP1368 and MAP1370 were constructed using the donor plasmids pMAP689, pMAP690, pMAP691, pMAP693 and pMAP695, resulting in the insertion of pglpABC, pglpD, pglpTQ or pglpFKX or plac fused with lacZ, respectively, into the galK locus in E. coli MDO.

Strains MAP1025, MAP1026, MAP1027, and MAP808 were constructed using the donor plasmids pMAP431, pMAP432, pMAP433, and pMAP205, respectively, resulting in the insertions PglpA, PglpD, PglpT, and PglpF, where the 16 bp upstream of the translation start site was changed to 5'-CAAGGA GGAAACAGCT -3' (SEQ ID NO: 10) fused to lacZ and inserted into the galK locus of E. coli MDO.

Strains MAPI176 to MAPI185 were constructed using donor plasmids pMAP486-pMAP495, respectively, resulting in inserts PglpF_SDl, PglpF_SD2, PglpF_SD3, PglpF_SD4, PglpF_SD5, PglpF_SD6, PglpF_SD7, PglpF_SD8, P glpF_SD9 and PglpF_SD10 fused to lacZ and inserted into the galK E loci. coli MDO. Modifications made to the ShineDalgarno sequence of the PglpF expression element are shown in FIG. 6A and are listed in table. 1.

Strains MAP1010-9, MAP1010-11, MAP1010-13, MAP1010-17, MAP1010-19, and MAP1010-20 were constructed using a plasmid preparation created using a degenerate primer. A modified PglpF expression element fused to lacZ was identified by sequencing and the promoter-lacZ fusion was inserted into the galK locus of E. coli MDO. Modifications made to the -10 region of the PglpF expression element are shown in FIG. 7A and are listed in table. 1.

Strains MAP1210, MAP1211, MAP1086, and MAP1209 were constructed using donor plasmids pMAP457, pMAP462, pMAP463, and pMAP537, which resulted in the insertion of a PglpF expression element with 25, 150, 175, or 190 bp removed from the 5′ end. Truncated versions of PglpF expression elements were fused to lacZ and inserted into the galK loci of E. coli MDO. Modifications of PglpF expression elements are listed in Table. 1.

Strain MAP1356 was constructed by double recombination as described by Sharan et al. (2009), Nat. Protoc., 4 (29:206-223). The glpR gene in MAP808 was replaced by a kanamycin resistance gene, resulting in MAP1356.

MAP700 and MAP710: strains were constructed using a helper plasmid and a donor plasmid containing nst cassettes (based on PglpF- or Plac-), resulting in E. coli MDO strains expressing N st -glycosyltransferase.

MAP219 and MAP986: strains were constructed using a helper plasmid and a donor plasmid containing Pd2 cassettes (based on PglpF- or Plac-), resulting in E. coli MDO strains expressing the Pd2 enzyme.

MDO1 and MDO15: Chemically competent Escherichia coli K-12 (DH1) MDO was transformed with 2 plasmids (Table 10) to produce LNnT and LNT, respectively. MP1497 - MP1499: Escherichia coli K-12 (DH1) MDO was transformed with the helper plasmid pACBSR and donor plasmids containing gene cassettes of interest (PglpF-lgtA or PglpF-galT) to integrate this glycosyltransferase into the genome. The resulting hosts were subsequently transformed with the appropriate plasmid carrying the antibiotic marker (Table 10).

MP166, MP245, MP1825, MP1920, MP2239, MP2374, MP2525: Escherichia coli K-12 (DH1) MDO was sequentially transformed with the helper plasmid pACBSR and donor plasmids containing gene cassettes of interest to ensure integration of the corresponding modifications into its genome in experiments using gene filling method (Table 10).

MAP265 and MAP425: Escherichia coli K-12 (DH1) MDO was transformed with the helper plasmid pACBSR and donor plasmids containing gene cassettes of interest to

- 56 044676 provide the possibility of introducing the desired inserts into the host genome by gene filling. The nadC gene was deleted by inserting galK into the nadC loci and by gene filling. Finally, the strains were transformed with plasmid pMAP101 to create strains MAP265 and MAP425.

MAP1200 and MAP1214: Escherichia coli K-12 (DH1) MDO was transformed with the helper plasmid pACBSR and donor plasmids containing gene cassettes of interest to allow the desired insertions to be introduced into the host genome by gene filling (Herring, CD., Glasner, J.D., Blattner , F.R. (2003), Gene (311), 153-163) (Table 10). All inserts were verified by sequencing (Eurofins Genomics, Germany).

Enzymatic assay: lacZ.

β-Galactosidase activity was assessed as described previously (see, for example, Miller JH Experiments in molecular genetics, Cold spring Harbor Laboratory Press, NY, 1972). Briefly, cells were diluted in Z-buffer and permeabilized with sodium dodecyl sulfate (0.1%) and chloroform. Analyzes were performed at 30°C. Samples were prewarmed, the assay was initiated by adding 200 μl of ortho-nitrophenyl-β-galactosidase (4 mg/ml) and stopped by adding 500 μl of 1 M Na ₂ CO ₃ when the sample became slightly yellow. The ortho-nitrophenol yield was subsequently determined as the change in absorbance at 420 nm. Specific activity is indicated in Miller units [A420/(min-ml-A600)]. Activities listed in table. 7 are averages from at least two independent experiments.

Enzymatic analysis: expression of heterologous genes Pd2 and Δ29nst.

Strains MAP219, MAP986, MAP700 and MAP710 were pre-cultured in 1 ml of basal medium (see above) supplemented with MgSOzj, thiamine and glucose at 34°C for 24 hours. 0.5 ml of the pre-culture was inoculated into 50 ml of minimal basal medium with the addition of MgSO ₄ , thiamine and 0.5% glycerol, and incubated with shaking at 28°C for 24 hours. 10 ml of cell cultures were collected in 50 ml centrifuge tubes at 8000xg, at -10°C for 15 minutes. The supernatant was removed and the cell pellet was stored at -80°C until use.

Cell pellets were resuspended in 1 ml physiologically similar 1-X medium (125 mM KCl, 25 mM _K3PO4 , 10 mM MSG, 0.001 mM _CaCl2 , 5 mM _MgSO4 , _pH 7.5) containing 1x BugBuster (Merk Milipore). Cell samples were lysed by ultrasound 4 times for 20 s at an amplitude of 30%. Insoluble cell debris was removed by centrifugation at 10,000xg for 10 min at -10°C. In the in vitro assay, 5 mM CMP-SA was used as the donor and 10 mM lactose was used as the acceptor. The donor, acceptor, and lysate were mixed, and samples were taken after 0, 5, 10, 20, and 30 min of reaction. Samples were boiled for 10 min and the supernatant was analyzed for 3′SL and 6′SL. Enzyme activity was measured in mM 6'SL or 3'SL production per hour and corrected for cell OD, and insertion of genetic cassettes was done by gene filling as described below, but could be done by any other method using homologous DNA recombination.

Analysis in deep well plates.

One colony from an LB plate was precultured in 1 ml of minimal basal medium containing glucose (0.5%) in 10 ml of a 24-well deep well plate (Deep well plate, Axygen). The plate was sealed before culture with Hydrophobic Gas Permeable Adhesive Seal (Axygen) and incubated for 24 h at 34°C with shaking at 700 rpm in an orbital shaker (Edmund Biihler GmbH). The cell density of the culture was monitored at 600 nm using an S-20 spectrophotometer (Boeso, Germany). 40 μl of the overnight culture was used for inoculation into 2 ml of minimal basal medium containing 0.01% glucose, 0.5% lactose, and 200 μg SUH (Sigma). IPTG was added as needed. Deep well plates were covered with sealing foil and incubated for 48 or 72 hours at 28°C in an orbital shaker at 700 rpm. After incubation, OD600 was measured and the plate was covered with heat sealing tape (Saveen Werner) and incubated in a thermomixer for 1 hour at 100°C with shaking at 400 rpm. The cell lysate was pelleted by centrifugation for 10 min at 4000 rpm. The concentration of HMO in the supernatant was determined by HPLC or HPAC methods.

Example 1: Cloning of 17 randomly selected regulatory elements isolated from E. coli.

To identify single-copy expression cassettes effective for expression in an E. coli host strain with E. coli K-12 DH1 chromosomal DNA, seventeen DNA fragments containing promoter elements were amplified. All promoter elements contained binding sites for transcription regulators, as well as an RNA polymerase binding site (regions -35, -10), transcription initiation sites, and a 5'-terminal untranslated sequence located 16 base pairs upstream of the translation initiation codon. A 16-nucleotide DNA sequence (5 - CAAGGAGGAAACAGCT-3) (SEQ id NO: 10) spanning the ribosome binding site (including the Shine-Dalgarno site) was introduced at the 3' end of all DNA fragments using sequence-specific oligonucleotides . The promoter names, promoter fragment lengths, and oligonucleotides used for amplification and insertion of SEQ ID NO: 10 are listed in Table. 11 below.

- 57 044676

Table 11

Promoter element Fragment length (bp), amplified moto s DH1 Name oligonucleotide eotide Oligonucleotide sequence SEQ ID NO 0350 ATGCGCAAAUCGGATCTCAAGGAA ATCGCAATGG SEQ NO:60 ID RaspV 334 0351 AGCTGTTUCCTCCTTGCTCATTGTC ATAGTGCGGGCAGG SEQ N0:61 ID 0354 ATGCGCAAAUGCTGAATCCGAACA CCAGCGTC SEQ NO:62 ID Pactp 134 0355 AGCTGTTUCCTCCTTGGCAGGACTT CATTATTAAGACGG SEQ NO:63 ID 0358 ATGCGCAAAUTACTCACTACTGAA ACAATATTGCC SEQ NO:64 ID PdcuB 584 0359 AGCTGTTUCCTCCTTGTAATCCTAT TTAAATTTTTGCTGAATAG SEQ NO:65 ID Pdps 182 0274 ATGCGCAAAUCCGAAAATTCCTGG CGAGCAG SEQ NO:66 ID 0275 AGCTGTTUCCTCCTTGGATGTTATG TCCCAGTAATTAAC SEQ NO:67 ID 0360 ATGCGCAAAUGAAGTAATCTTTCT TCACCTGCGTTC SEQ NO:68 ID PgalP 414 0361 AGCTGTTUCCTCCTTGGTTATTTTT TATTGTGAATTAAGATAGG SEQ NO:69 ID 0265 ATGCGCAAAUCAGTTCTTCTGCCG AAGGTT SEQ NO:70 ID PgapA 355 0266 AGCTGTTUCCTCCTTGTTGTTAGTG AATAAAAGGTTGCC SEQ N0:71 ID 0378 ATGCGCAAAUGAAAACATTCATAA ATTAAATGTG SEQ NO:72 ID PglpA 166 0379 AGCTGTTUCCTCCTTGTTCGTTTTTT ACCATTTAGCCATAG SEQ NO:73 ID 0376 ATGCGCAAAUGCGTCTCTCTTTCTT TASAAAAS SEQ NO:74 ID PglpD 173 0377 AGCTGTTUCCTCCTTTGTTCGTTAAA GCTCATAAATGTTCG SEQ NO:75 ID 0261 ATGCGCAAAUGCGGCACGCCTTGC AGATTACG SEQ NO:76 ID PglpF 284 0262 AGCTGTTUCCTCCTTGGTTAATGTT TGTTGTATGCG SEQ NO:77 ID

- 58 044676

PglpT 229 0380 ATGCGCAAAUCCATTTAGCCATAG TAAAACATG SEQ NO:78 ID 0381 AGCTGTTUCCTCCTTGCCGTGGTCT TATTTATTGATTAAC SEQ NO:79 ID ATGCGCAAAUGCGCGGGGTTCCGTG SEQ ID 0270 CGTGGG NO:80 AGCTGTTUCCTCCTTGATTTATTAC SEQ ID PkatE 254 0271 TGAAAGGGGCCGC NO:81 ATGCGCAAAUGTGATCAAAATTT SEQ ID 0272 TAAACAG NO:82 AGCTGTTUCCTCCTTGACAGTGTTA SEQ ID PkatG 254 0273 CCGTTACGATAC NO:83 ATGCGCAAAUTGTGAGTTAGCTCA SEQ ID 068 CTCATTAG NO:84 AGCTGTTUCCTCCTTGAAATTGTTA SEQ ID Plac 91 0268 TCCGCTCACAA NO:85 ATGCGCAAAUGAATGCCTCAGGT SEQ ID 0257 GAGGG NO:86 AGCTGTTUCCTCCTTGTTTCGCGCT SEQ ID Pmlc 134 0258 CCGAAATAATC NO:87 ATGCGCAAAUCCGAAATCGCTGAA SEQ ID 0366 GGTTACGTAC NO:88 AGCTGTTUCCTCCTTGAATGTGATA SEQ ID PpoxB 184 0367 ACGGTAACAAGTTTAG NO:89 ATGCGCAAAUGGCTGTGTTGAAAG SEQ ID 0255 GTGTTG NO:90 AGCTGTTUCCTCCTTGAGTATGGGT SEQ ID pptsG 384 0256 GCTTTTTTTACG NO:91 ATGCGCAAAUGAATTGCAACAGTA SEQ ID 0259 ATGCCAG NO:92 AGCTGTTUCCTCCTTGATAGGTTTA SEQ ID PptsH 382 0260 GTGTTGTGGAAC NO:93

Of the seventeen DNA fragments cloned (17 promoters), all identical at the 3' end (SEQ ID NO: 10), all were fused to the promoterless lacZ gene linked to a transcription terminator sequence, T1. A single copy of the PromoterSEQ ID NO:10-lacZ-T1 expression cassette was integrated into chromosomal DNA. The expression activity of the lacZ gene introduced in a single copy was measured as β-galactosidase activity. lac promoter activity was measured in the presence of IPTG. The results of reporter gene expression from the constructs are shown in FIG. 1A. Notably, not all promoters tested showed an increase in activity upon replacement of the native 16 nucleotide fragment upstream of the translation initiation codon containing the ribosome binding site (RBS) for SEQ ID NO: 10. Remarkable activity was observed for the glpF promoter, activity for the glpA and D promoters was also significant, but the activity of most promoters tested either did not change at all or did not increase significantly (as would be expected according to Meynial-Salles I. et al. (2005), Appl. Eviron. Mcrobiol., 71:2140- 2144; and WO 03/08960). FIG. 1B shows reporter gene expression data from constructs comprising three representative promoters from FIG. 1A (PglpF, PglpA and PglpT) and any native 16 nucleotide sequence derived from the 5'UTR of DNA upstream of the corresponding gene (i.e. glpF, glpA and glpT), or SEQ ID NO: 10.

In fig. Figure 2 shows a comparison of reporter gene expression levels with promoter sequences isolated from the glp operons: glpFKX, glpABC, glpTQ and glpD. All promoters are negatively regulated by the GlpR repressor. All cloned DNA fragments contained DNA binding sites for

- 59 044676 cAMP-CRP plus a number of binding sites for other regulatory elements, such as one or more GlpR repressor binding sites. A schematic view of the cloned promoter elements is shown in FIG. 2 and 3. Cloning was performed as described above.

Example 2. Expression level from a single copy of Pglp-lacZ fusion constructs.

A promoter plasmid containing the lacZ gene without a promoter was used as a cloning system to identify E. coli promoter elements that could support high and regulated protein expression. Levels of lacZ expression were determined from both a single copy of Pglp-lacZ integrated into chromosomal DNA and from a high copy number plasmid (as described in Example 11). The ΔlacZM15 deletion in the lacZ gene in E. coli MDO fails to produce active β-galactosidase enzyme and was therefore used as a background strain in the screen. The Plac promoter element was used as a positive reference for lacZ expression. Promoter elements derived from the glp, glpTQ, glpACB, glpD and glpFKX, or Plac operons were fused to the promoterless lacZ gene and inserted into the Escherichia coli genome by site-specific recombination, resulting in strains MAP1367, MAP1365, MAP1366, MAP 1368 and MAP 1370 respectively. All isolated promoter DNA fragments, pglpTQ, pglpACB, pglpD, pglpFKX and plac fused to lacZ, can express β-galactosidase enzyme, and enzyme activity was measured as shown in FIG. 4. It should be noted that Plac promoter activity was measured in the presence of IPTG.

In another set of experiments, a 16-bp nucleotide sequence located upstream of the translation start sites in the glpF, glpA, glpD, glpT, and lacZ genes included in expression cassettes containing pglpFKX, pglpACB, pglpD, and pglpTQ was replaced by the sequence 5'-CAAGGAGGAAACAGCT- 3' (seq id no: 10), resulting in strains MAP808, MAP1025, MAP1026 and MAP1027, respectively. Modification of 16 base pairs upstream of the translation start site in constructs containing the PglpF, PglpA and PglpT promoter elements and the original 5'UTR DNA fragment of the corresponding glp gene increased the expression of the β-galactosidase enzyme approximately 10,000-fold for PglpF, 2-fold for PglpA and PglpT (Fig. 4). Surprisingly, an additional replacement of the original 54 nucleotide 5'UTR DNA sequence located downstream of the transcription start point in the PglpA and PglpT constructs to SEQ ID NO: 36 of the 5'UTR DNA of the glpF gene (resulting PglpA_70UTR DNA constructs (SEQ ID NO: 50 ) and PglpT 70UTR (SEQ ID NO: 51)), gave a significant increase in reporter gene expression from these constructs (Fig. 4), demonstrating the unexpected synergistic effect of the 70UTR sequence on gene expression.

Table 12

Primers used to construct PglpA_70UTR and PglpT_70UTR

PglpA_ 70UTR 037 8 ATGCGCAAAUGAAAACATTCATAAATTAAATGTG SEQ ID NO:94 081 2 AGCTGTTUCCTCCTTGGTTAATGTTTGTTGTATGCGT GAAAGTCACGGACCTCCACGATGCTTGTAGGCATCG CGCATATTCGCTCATAATTC SEQ ID NO:95 PglpT. 70UTR 038 0 ATGCGCAAAUCCATTTAGCCATAGTAAAAACATG SEQ ID NO:96 081 5 AGCTGTTUCCTCCTTGGTTAATGTTTGTTGTATGCGT GAAAGTCACGGACCTCCACGATGCTTGTAGGCATGC CGCGATGTTAAGAAAAC SEQ ID NO:97

Example 3. The level of expression from a single copy of Pglp-lacZ fusion constructs is catabolically suppressed.

Since expression from the glp promoters is known to be catabolically repressed, the level of expression from Pglp-lacZ fusion constructs containing SEQ ID NO: 10 was measured. Strains MAP808, MAP1025, MAP1026 and MAP1027 were grown in the presence or absence of glucose, and β- activity was determined. galactosidase encoded by lacZ (Table 13). In both LB and minimal medium, expression levels were significantly reduced in the presence of glucose.

- 60 044676

Table 13

Levels. expression from single copies of Pglp-lacZ fusion constructs

Strain Promoter element LB (MU) LBglucose (MU) MAR8O8 PglpF 8.850±l, 415 279±3 MAR102 5 PglpA 3.732±23 4 72±70 MAR102 6 PglpD 3.969±60 3 618±223 MAR102 7 PglpT 647±23 15±15

Degree of suppression of scientific research (times) MMglycerin MMglucose Degree of suppression (times) 32 13,450 ±786 3.699±35 3 4 51 5.677±154 1.874±33 2 3 6 4.529±132 1.398±12 1 3 43 1.110±139 290±44 4

Example 4. Expression from a single copy of PglpF-lacZ is regulated and depends on the carbon source in the carrier.

To determine whether the expression level of PglpF-lacZ is regulated by carbon sources other than glucose, strain MAP808, which contains a single copy of the PglpF-lacZ fusion construct, was grown in LB medium with and without glucose, and in minimal medium using glycerol, sorbitol , maltose or glucose as the sole carbon source. High levels of lacZ expression were observed in LB medium, but the addition of 0.2% glucose significantly reduced lacZ expression (32-fold) in LB medium (Table 13, Fig. 5). High levels of lacZ expression in strain MAP808 were observed in minimal media when glycerol or sorbitol was used as the sole carbon source, whereas the use of maltose or glucose in minimal media reduced lacZ expression.

Example 5 The level of expression in the PglpF and T constructs can be significantly changed by changing the ribosome binding site contained in SEQ ID NO: 10.

Effect of modifications to the recombinant sequence (SEQ ID NO: 10) containing the ribosome binding site (Shine-Dalgarno sequence) in constructs containing PglpF and PglpT (SEQ ID NO: 54 and 49), and the 70UTR sequence (SEQ ID NO: 37) gene expression was determined by measuring the expression level of a reporter gene (lacZ) expressed from various PglpF and PglpT constructs containing variants of SEQ ID NO: 10, as shown in FIG. 6. The expression level of lacZ changed significantly (decreased by 9-95% or increased by 60%) (Fig. 6).

Example 6 The expression level of PglpF can be changed by changing the sequence of the -10 region.

Nucleotide modifications were introduced into region-10: 5'-TAAGT-3' of the 310 bp PglpF expression element, resulting in strains MAP1010-19, MAP1010-20, MAP1010-13, MAP1010-17, MAP1010-11 and MAP1010- 9. The expression level of lacZ was measured in each strain, and the results showed that lacZ expression was reduced by 16–60%, showing the importance of region-10 in changing expression levels (Fig. 7).

Example 7 The 120 bp fragment containing PglpF contains a functional promoter.

The activity of PglpF was examined by truncation by 15, 140, 165, or 180 bp from the 5' end, resulting in strains MAP1210, MAP1211, MAP1086, and MAP1209, respectively. The expression level of lacZ was measured in each strain, and the results showed that expression with PglpF was maintained even when the PglpF sequence length was reduced from 300 bp to 120 bp (Fig. 8).

Example 8: Removal of the transcriptional repressor GlpR increases expression from the PglpF element.

The PglpF promoter is known to be negatively regulated by the transcriptional repressor protein GlpR. The 300-bp PglpF contains four binding sites for the repressor protein GlpR (Fig. 2). To determine the importance of the GlpR repressor on transcription from PglpF, the glpR repressor gene was knocked out, resulting in strain MAP1356. The expression level of lacZ was measured in the absence and presence of the repressor GlpR, and the expression level increased by almost 20% in its absence (Fig. 9).

Example 9 Expression of recombinant genes using the PglpF expression element depends on the carbon source used in the growth medium.

Growing cells in different media has a significant effect on the level of LacZ expressed with PglpF (Fig. 5). To test whether heterologous genes can be expressed from the PglpF promoter element linked to the 70UTR DNA sequence and whether expression is affected by

- 61 044676 carbon source used in the medium, strains MAP710 and MAP986 were constructed expressing nst or Pd2 transsialidase with a single copy of PglpF, respectively, and cultured in minimal media containing glucose or glycerol as a carbon source. When glycerol was used as a carbon source, enzymatic activity was at least 7 times higher than when cells were grown on media containing glucose as a carbon source (Fig. 10), showing that recombinant genes can be expressed using PglpF and that this expression can be regulated by the carbon source present in the medium.

Example 10. High level of expression of recombinant genes using the PglpF expression element compared to using the Plac promoter element.

The expression of heterologous genes from the fusion constructs PglpF-70UTR-Pd2 and PglpF-70UTR-nst was determined for single copies integrated into the host chromosome, followed by measurement of the enzymatic activity of the expressed proteins. Enzyme activity was measured in cell lysates expressing Pd2 from the Plac promoter (induction with IPTG) or from the PglpF promoter (Fig. 10). 6′SL production was 7-fold higher in cell lysates expressing Pd2 from PglpF compared to Plac. Similarly, the enzymatic activity of cell lysate expressing nst siallyltransferase from the Plac promoter (inducible by IPTG) or from the PglpF promoter (Fig. 10) showed almost 14-fold higher activity when nst was expressed with PglpF than with Plac.

Example 11 Pglp-containing nucleic acid constructs can be used for expression in high copy number plasmids.

The PglpF, PglpA and PglpT nucleic acid constructs all contain a promoter DNA fragment isolated from the respective glp operons and a synthetic DNA sequence containing the 5'UTR DNA sequence isolated from the corresponding promoter, which has been fused to SEQ ID NO: 10 and placed upstream of the translation start site of the reporter gene (lacZ). The constructs were cloned into a high copy number plasmid. lacZ expression levels were measured as β-galactosidase enzyme activity in cells grown in LB medium in the presence or absence of glucose (Table 14, Fig. 11). Results in table. 4 show that Pglp maintains high expression from high copy number plasmids and that this expression is regulated by catabolic repression in the presence of glucose (Table 14, Fig. 11).

Table 14

Expression levels from promoter-lacZ fusion constructs in a high copy number plasmid

Plasmid Promoter element Original operon Activity in LB (MU) Activity in LB+glucose (MU) pMAP205 PglpF glpFKX 51.245±4.560 6,664 pMAP431 PglpA glpACB 27.377±549 457±469 pMAP433 PglpT glpTQ ll.019±575 400±10

Example 12: Engineering of Escherichia coli to produce LNnT using the PglpF promoter.

Escherichia coli strain K-12 (DH1) MD0 can be modified to sporadically express heterologous genes of interest. For example, strain MDO1 is a 2-plasmid strain with a medium copy number plasmid (30-40 copies per cell) carrying the lgtA gene and a high copy number plasmid (300-500 copies per cell) carrying the galT gene. The Plac promoter controls the expression of both heterologous genes in these plasmids. Alternatively, heterologous genes can be integrated into the genome of Escherichia coli strain K-12 (DH1) MDO to create genome-derived production systems. Thus, the mid-copy plasmid with the lgtA gene is replaced by one genomic copy of the PglpF-lgtA expression cassette in strain MP1497, which still carries the high-copy plasmid with Plac-galT. In another example, the high-copy galT gene plasmid is replaced with one genomic copy of the PglpF-galT expression cassette in strain MP1499, which still carries the mid-copy Plac-lgtA plasmid. As shown in FIG. 12A, similar LNnT titers are achieved when expression of both heterologous genes, lgtA and galT, is expressed from the plasmid Plac promoter (strain MDO1) or when one of the genes is expressed from a fragment with the PglpF promoter integrated into chromosomal DNA and the second gene is expressed from the plasmid Plac promoter (strains MP1497 and MP1499).

Strains MP2622 and MP1825 express the lgtA and galT genes from one genomic copy under the control of the Plac or PglpF promoters, respectively. Strain MP166 has three Plac-controlled genomic copies of both genes. With the exception of strain MP2622, the lacI gene is removed from the genome of the strains discussed herein, and the CP6-galK cassette is inserted into the lacI locus. From the histograms in Fig. 12V

- 62 044676 it is obvious that the expression of the lgtA and galT genes from a single PglpF-controlled genomic copy (strain MP1825) is sufficient to achieve LNnT titers, which are significantly higher than the titers achieved with single (strain MP2622) or multiple (strain MP166) Plac- controlled genomic copies of lgtA and galT integrated into the genome.

Example 13: Engineering Escherichia coli to produce LNT using the PglpF promoter.

Escherichia coli strain K-12 (DH1) MDO can be modified to sporadically express heterologous genes of interest. Strain MDO15 is a 2-plasmid strain with a medium copy number plasmid (30-40 copies per cell) carrying the lgtA gene and a high copy number plasmid (300-500 copies per cell) carrying the galTK gene. The expression of both heterologous genes in these plasmids is controlled by the Plac promoter. Alternatively, heterologous genes can be integrated into the genome of Escherichia coli strain K-12 (DH1) MDO to create genome-wide production systems. Thus, the medium copy number plasmid with the lgtA gene is replaced by one copy of the PglpF-lgtA expression cassette in strain MP1498, which still carries the high copy number plasmid with Plac-galTK (Fig. 13A). In another example, the high copy number plasmid with the galTK gene is replaced by two genomic copies of the PglpF-galTK expression cassette in strain MP1655, which still carries the medium copy number plasmid with Plac-lgtA (Fig. 13A). As shown in FIG. 13A, similar LNT titers can be achieved when expression of both heterologous genes, lgtA and galTK, is controlled by the plasmid Plac promoter (strain MDO15), or when lgtA expression is driven by a PglpF-lgtA cassette inserted into the host chromosomal DNA and the plasmid Plac promoter controls expression galTK gene (strain MP1498). Similar product titers can also be achieved when two genome-integrated PglpF-lgtA cassettes drive galTK gene expression and lgtA expression is under the control of the plasmid Plac promoter (strain MP1655 in Fig. 13A).

The expression of three genomic copies of lgtA and two galTK in strain MP245 is under the control of the Plac promoter. One copy of each heterologous gene is expressed from the PglpF promoter in strain MP1920. The lacI gene is removed from the genome of these strains by introducing a Cpb-galK cassette into the lacI locus. From the graphs in Fig. 13B it is obvious that the expression of the lgtA and galTK genes from a single PglpF-controlled genomic copy (strain MP1920) achieves LNT titers that are much higher than those achieved in the variant when multiple genomic copies of lgtA and galT controlled by Plac (strain MP245) are integrated into a chromosome.

Example 14: Engineering Escherichia coli to produce LNFP-I using the PglpF promoter.

A genomic LNFP-I production system can be developed by integrating the corresponding heterologous genes into the genome of Escherichia coli strain K-12 (DH1) MDO. For example, strain MP2239 expresses the heterologous genes lgtA, galTK and futC from one genomic copy under the control of the PglpF promoter, while the expression of the colanic acid (CA) genes is gmd, wcaJ (fcI), wcaH (gmm), wcaI, cpsB (manC) and cpsG (manB) - controlled by Plac. Integration of extragenomic copies of genes for the colanic acid synthesis pathway in strain MP2239 under the control of the PglpF promoter produces strain MP2374. The lacI gene is removed from the genetic background of both strains. A marked improvement in LNFP-I titer was observed when additional copies of colanic acid synthesis pathway genes were integrated into strain MP2239 and expressed from the PglpF promoter (Fig. 14).

Example 15: Engineering Escherichia coli to produce 3'SL using the PglpF promoter.

In strain MAP425, the Plac promoter was used to express i) the heterologous nst gene and the heterologous neuBCA gene cluster integrated into the Escherichia coli K-12 (DH1) MDO chromosome; and ii) a cluster of heterologous neuBCA genes from a high copy number plasmid (300-500 copies per cell). In strain MAP1214, the PglpF promoter was used to express heterologous genes nst, neuA, neuB and neuC, integrated in single copies into the Escherichia coli K-12 (DH1) MDO chromosome. As shown in Figure 15, similar 3'SL titers were achieved when Plac was used to express the neuBCA gene cluster from a high copy number plasmid (strain MAP425), and when PglpF was used to express nst, neuA, neuB and neuC from single integrated copies of the genes ( strain MAP1214) (Fig. 15).

Example 16: Engineering Escherichia coli to produce 6'SL using the PglpF promoter.

In strain MAP265, the Plac promoter was used to express i) the heterologous Pd2 gene introduced into the Escherichia coli K-12 (DH1) MDO chromosome, and ii) the heterologous neuBCA gene cluster from a high-copy plasmid (300-500 copies). In strain MAP1200, a fragment of the PglpF promoter was used to express the heterologous genes Pd2, neuA, neuB and neuC, integrated in single copies into the Escherichia coli K-12 (DH1) MDO chromosome. As shown in FIG. 16, the 6'SL titer was significantly higher when PglpF was used to express Pd2, neuA, neuB and neuC from single integrated gene copies than when Plac was used to express the neuBCA gene cluster from a high copy plasmid (strain MAP265) (Fig. 16).

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Claims (16)

Example 17: Engineering Escherichia coli to produce 2'FL using the PglpF promoter. Strain FT18 is a 2-plasmid strain with a medium copy number plasmid (30-40 copies per cell) carrying the colanic acid biosynthetic pathway genes gmd, wcaJ (fcI), cpsB (manC) and cpsG (manB) isolated from Escherichia coli K-12 DH1 and a high-copy plasmid (300-500 copies per cell) with the futC gene. The Plac promoter controls the expression of cloned genes in these plasmids. Expression of the futC genes and the colanic acid biosynthesis genes gmd, wcaJ (fcI), cpsB (manC), and cpsG (manB) in strain MP965 is under the control of the PglpF promoter. A single copy of the futC and colanic acid genes is expressed from the PglpF promoter in strain MAP965. The results presented in Fig. 17 show that expression of the futC and CA genes from the colanic acid biosynthesis genes from a single PglpF-controlled genomic copy (strain MAP965) allows one to obtain almost the same 2TL titers as when expressed from multiple copies of the Plac-controlled genes (Fig. 17) . CLAIM 1. A nucleic acid construct suitable for modifying the expression of a desired gene, comprising a synthetic non-coding DNA sequence (i) that contains a first DNA fragment and a second DNA fragment, wherein the first DNA fragment is a DNA sequence derived from the 5' untranslated region (5 '-UTR) of the glpF, glpT, glpA or glpD gene from Escherichia coli, and the second DNA fragment is the DNA sequence CAAGGAGGAAACAGCT (SEQ ID NO: 10) or a variant of the said sequence selected from the DNA sequences SEQ ID NO: 38-47, and where the first fragment is located above the second fragment. 2. The nucleic acid construct according to claim 1, further comprising a promoter DNA sequence (ii), where the promoter DNA is operably linked to the synthetic non-coding DNA sequence (i) and is located before the first DNA fragment of the synthetic non-coding DNA sequence (i). 3. The nucleic acid construct according to claim 1 or 2, further comprising a coding DNA sequence (iii), where the coding DNA sequence (iii) is operably linked to a synthetic non-coding DNA sequence (i) and is located downstream of a second fragment of the synthetic non-coding DNA sequence (i ). 4. The nucleic acid design according to claim 1, 2 or 3, wherein the first DNA fragment of the synthetic non-coding DNA sequence (i) is derived from the 5'UTR DNA sequence of the glpF, glpA or glpD gene of Escherichia coli. 5. The nucleic acid construct of claim 1, 2 or 3, wherein the first DNA fragment of the synthetic non-coding DNA sequence (i) contains from about 5 to about 65 contiguous nucleotides after the start of transcription of the glp gene, wherein preferably the glp gene is glpF. 6. The nucleic acid construct of any one of the preceding claims, wherein the first DNA fragment of (i) is or contains SEQ ID NO: 36, a fragment thereof. 7. The nucleic acid construct of any one of the preceding claims, wherein the synthetic non-coding DNA sequence (i) is SEQ ID NO: 37. 8. The nucleic acid construct according to any one of claims 2 to 7, wherein the promoter DNA sequence (ii) is derived from a promoter DNA sequence that naturally initiates transcription of the Escherichia coli glpFKX, glpABC, glpD or glpTQ operon. 9. The nucleic acid design according to any one of claims 2 to 8, wherein the promoter DNA sequence (ii) is selected from SEQ ID NO: 48, 49 or 54. 10. The nucleic acid construct of any one of the preceding claims, which is selected from any of the nucleotide sequences specified in SEQ ID NO: 12-35, 50-53, or it is a fragment or variant of the specified sequence. 11. The nucleic acid construct of any one of the preceding claims, wherein the DNA coding sequence (iii) encodes a protein or RNA, wherein preferably the protein or RNA has a function that is necessary or useful for the production of one or more human milk oligosaccharides (HMOs) in a recombinant organism . 12. The nucleic acid construct of any one of the preceding claims, wherein the construct is an expression cassette that is integrated into the genomic or plasmid DNA of a recombinant organism. 13. A recombinant cell containing one or more nucleotide constructs according to any one of claims 1 to 12. 14. The recombinant cell according to claim 13, wherein the cell is a bacterial cell. 15. The recombinant cell according to claim 14, wherein the bacterial cell is E. coli. 16. An expression system containing a nucleotide construct according to any of claims 1-12 or a recombinant cell according to any of claims 13-15. -